BIOL2174 Cell Physiology Lecture Notes PDF

BIOL2174: Cell Physiology – Lecture Notes Lecture 1 What is cell physiology: Physiology – study of function of living systems Structure and function of cell membranes, contractile systems, cellular organelles, membrane channels, transporters, and pumps Integrated re...

BIOL2174: Cell Physiology – Lecture Notes Lecture 1 What is cell physiology: Physiology – study of function of living systems Structure and function of cell membranes, contractile systems, cellular organelles, membrane channels, transporters, and pumps Integrated regulation of cellular function, including mechanisms of signal transduction, development, gene expression, cell-to-cell interactions, and the cell physiology of pathophysiological states Example 1 – Multidrug resistant cancer Cancer cells display several drug-resistance mechanisms One involves increased levels of ATP-driven pump in the cell’s plasma membrane This pump (P-glycoprotein) can move a wide variety of anti-cancer drugs out of cancer cells, thereby preventing their accumulation in the cell cytosol This pump is a member of a very large family of membrane proteins called the ABC transporters There is interest in identifying inhibitors of this pump, that may be used in the clinic to reverse multidrug-resistance Example 2 – Food poisoning The woman’s condition was actually due to severe tetrodotoxin poisoning after eating roe of the puffer fish It was fortunate that appropriate aggressive resuscitation was instituted to revive the patient from her critical state Tetrodotoxin reversibly blocks a Na+ channel, the opening of which triggers the propagation of the action potential in excitable (never and muscle) cells Example 3 – Insensitivity to pain We have many types of neurons and 9 types of Na channels Patients with congenital insensitivity to pain either lack of have mis-functional Nav1.7 (the Na channel used to pick up an initial pain signal) Intentionally block Na channels – local anaesthetic – Nav1.7 now a major target for pain killing medication Example 4 – Malaria Spiroindolones (a potent compound class for the treatment of Malaria) – identified in a screen of 12,000 natural products and related compounds The spiroindolones kill malaria parasites at low concentrations On prolonged exposure of parasites to spiroindolones resistant parasites emerge Resistance is due to mutations in a protein called PfATP4 PfATP4 is on the parasite plasma membrane Resistance to cipargamin (now in phase III clinical trials) has emerged during trials BIOL2174: Cell Physiology – Lecture Notes Lecture 2 – Simple Diffusion across membranes Biological membrane structure Polar head – hydrophilic (water loving) Non-polar tail – hydrophobic (water fearing) 1. Dissolving into the membrane – dependent on drug hydrophobicity 2. Diffusing through the interior of the membrane – dependent to drug size Factors affecting simple diffusion across membranes We can understand the rate at which solutes diffuse across the lipid component of membranes in terms of the need for them to: o Partition – from aqueous phase into the oily interior of the membrane bilayer, as reflected in their oil/water partition coefficient (K) o Diffuse – from one side of the bilayer to the other, as reflected in the size – dependence of the diffusion rate BIOL2174: Cell Physiology – Lecture Notes As a result, the rate of small solutes through the lipid phase of biological membranes is determined largely by its hydrophobicity (which determines its oil/water partition coefficient) and its size (which determines its rate of diffusion within the membrane bilayer) The basal membrane permeability (P), increases with increasing solute hydrophobicity and decreases with increasing solute size Polar and non-polar compounds Polarity refers to the distribution of electric charge around atoms or molecules Polar molecules occur when there is uneven sharing of electrons caused by electronegativity differences between bonded atoms Non-polar molecules occur when electrons are charged equally, or when bonds in molecules cancel each other out Diffusion across membranes Accumulation of very hydrophobic drugs in membrane BIOL2174: Cell Physiology – Lecture Notes Examples of the importance of simple diffusion in a clinical context 1. The antimalarial action of chloroquine Chloroquine is a weak base The malaria parasite has an acidic ‘digestive vacuole’ Chloroquine accumulates in the vacuole by ‘weak base trapping’ BIOL2174: Cell Physiology – Lecture Notes Summary – chloroquine delivery The delivery of CQ at high concentrations to its site of action within the parasite is dependent upon: 1. The ability of the uncharged, hydrophobic CQ molecule to diffuse through the membranes to reach the interior of the food vacuole 2. The inability of the charged (and therefore hydrophilic) form of the CQ to escape from the acidic vacuole 2. Morphine Isolated – 1805 Commercialised – 1827 Strong pain suppressant acting on the central nervous system No. 2 = codeine No.3 = heroin → least polar, more permeable, fastest onset of action, most potent Ester trapping of drugs o Greater membrane permeability = rapid onset o Accumulation in cell = greater potency BIOL2174: Cell Physiology – Lecture Notes 3. Treatment of an inherited disorder Hartnup disease o An autosomal recessive condition characterised by defective uptake of neutral amino acids in the gut and the kidney (i.e., it is a defect in a membrane transporter) o One of the neutral amino acids, tryptophan is a precursor for the neurotransmitter serotonin o Symptoms – dermatitis, diarrhea, ataxia, neuropsychiatric symptoms Case study o 3yo child o Growth failure, developmental delay, hyperactivity, chronic diarrhea, weakness o The child does not take up dietary (i.e., oral) tryptophan (but serum levels do rise if the tryptophan is injected into the blood stream) BIOL2174: Cell Physiology – Lecture Notes Hartnup disease – case study o The graph shows that when the child was given tryptophane ethyl ester orally, at either a high dose, there was an increase in the serum tryptophan levels. This contrasts with response to oral tryptophan o The next graph shows the improvement in the child’s growth following the inclusion of (low dose) tryptophane ethyl ester in the diet BIOL2174: Cell Physiology – Lecture Notes Lecture 3 – Bioelectricity and cellular driving forces Driving Force 1: Concentration gradient Molecules can be driven to move across the cell membrane due to different concentrations on each side Molecules will move from a high concentration to low concentration until an equilibrium is reached due to random motion o Electrochemical equilibrium (balancing electrical force) Equilibrium potential E.g., there is a driving force to move Na+ from outside to inside the cell Driving Force no. 2: Membrane potential An electric potential difference across the membrane will drive charged particles to try cross the membrane Electrical potential is created by different numbers of charged particles on each side Ions can still move across the membrane at electrochemical equilibrium but the number coming in = number going out Ion concentration is different inside and outside a cell because of pumps (primary active transport), transporters (secondary active transport), ion selective channels The membrane can be made selectively permeable to one ion type by the use of ion selective channels Summary: Diffusion – molecules will try to move across the membrane if they are at different concentration on each side Membrane potential – an electric potential difference across the membrane will drive charged particles to try to cross the membrane o The membrane potential is created by different no. of charged particles on each side of the membrane BIOL2174: Cell Physiology – Lecture Notes How membrane potentials are calculated: Equilibrium potential for Na+ (ENa+): o The potential inside the cell compared to outside if Na+ was the only permeable ion o The inside of the cell will become more positive compared to outside the cell because when Na comes across the inside will become more positive because ti brings its +ve charge, leaving the outside more negative If concentrations change the equilibrium potential will too BIOL2174: Cell Physiology – Lecture Notes Cell resting potential How to calculate membrane potential because in reality the membrane is a bit permeable to all ion types The most permeant ions will have a big effect on the membrane potential The least permeant ions will have only a small effect All cells at rest have a -ve membrane potential Cell resting potential will occur when there is no net charge movement across the membrane Fluxes that make the cell more -ve are balanced by those making it more +ve Why cells need a membrane potential: A cell can change membrane potential very quickly by changing the permeability of the ions by opening ion channels BIOL2174: Cell Physiology – Lecture Notes Making use of the electrochemical potential Moving ions down the electrochemical potential releases energy that can be used by the cell Similarly moving things against the electrochemical potential takes energy o E.g., generating ATP synthase How much work can the electrochemical potential do Membrane potentials recap Channelopathy Disease caused by defective ion channels Usually changes either channel permeability of voltage sensitivity Myotonia congenita BIOL2174: Cell Physiology – Lecture Notes Congenital – present from birth Over 80 different mutations that can cause the disorder – usually chloride channels Symptoms o Delayed relaxation of skeletal muscle after contraction o Spontaneous paralysis Causes o Changes in membrane potential o +ve ion influx → membrane depolarisation (action potential) → muscle contraction o Influx of Cl- (& K+) ions to recover after membrane depolarisation (repolarisation) o Therefore, Cl channels are necessary for rapidly resetting the membrane potential after a stimulus ▪ Mutations that reduce Cl- movement cause delayed repolarisation BIOL2174: Cell Physiology – Lecture Notes Lecture 4 – Ion Channels Why do we need membrane transport proteins Many molecules that are essential to the life of a cell cannot cross the membrane without assistance Ways to cross the membrane Membrane transport proteins Channel – a protein which when ‘open’ provides an aqueous pathway that allows for the diffusion of solutes from one side of the membrane to the other o Has a continuous pore (when open) o Can move millions of ions per second Transporter – a protein which has a solute binding site and which undergoes a conformational change in order to allow the passage of the solute form one side of the membrane to the other o Never has a continuous pore o Can move about 1000 ions per second Channels vs transporters BIOL2174: Cell Physiology – Lecture Notes Types of channels Passive vs active transport Passive transport – solutes move down an electrochemical gradient Active transport – energy is required to move solutes against their electrochemical gradients Hodgkin & Huxley (1950s) Ion currents in Squid axon Were able to show that the action potential was due to the movement of ions through separate pathways they called ion channels Sodium ions to contribute to action potential o Test the theory that action potential propagation has a dependency for Na+ BIOL2174: Cell Physiology – Lecture Notes o Varying [Na+] in solution o Measure membrane potential in response Two ways to measure the electrical properties of a cell 1. Membrane potential measures a. Apply a stimulus/shock/current b. Measure the membrane potential (voltage of the cell) 2. Voltage clamp (current measurement) a. Fix the membrane potential b. Measure the current flowing across the membrane (i.e., through channels) Inward and outward currents Negative current – inward o Inward movement of +ve ions – or an outward movement of -ve ions o E.g., Na+ moving into a cell Positive current – outward o Outwards movement of +ve ions – of an inward movement of -ve ions o E.g., K+ moving out of a cell Hodgkin & Huxley cont. Depolarisation produced a large biphasic current They were able to show that this complex current was due to the inward current of Na+ and the outward current of K+ through separate pathways Separating current components with ion substitutions BIOL2174: Cell Physiology – Lecture Notes o Replace Na+ with choline or K+ with Cs+ Separate pathways proved by channel blockers o Tetrodotoxin selectively blocks Na channels Hyperpolarisation produces negligible inward current Ion currents in Squid Axon summary Invented the voltage clamp method for studying cellular currents Showed the action potential is due to the inward motion of Na+ followed by the outward motion of K+ Hypothesised that this occurred through separate selective pathways they called ion channels, that respond to changes in membrane potential (voltage-gated ion channels) They developed a mathematical model to describe the action potential and its propagation along the axon, based upon these 2 channel types BIOL2174: Cell Physiology – Lecture Notes Lecture 5 – Ions Channels & Action Potentials Proof of channels from Single channel measurements Current passing through a single Na channel How long does it take for a channel to open or close o Roughly milliseconds How many ions pass through the channel per second o Millions of ions Patch clamp recording modes Cell Attached Mode – used to measure the current passing through a single ion channel Inside Out Mode – used to measure the current passing through a single ion channel Whole Cell Mode – used to measure the current passing through membrane transporters Outside Out Mode Characteristics of the channel BIOL2174: Cell Physiology – Lecture Notes This is because Na channels inactivate Voltage gated Na+ channel Can exist in 3 states o Closed o Open o Inactivated Action (voltage) gate o Voltage moves channel between o Closed → open (depolarisation) o Inactivated → closed (repolarisation) Inactivation gate o Voltage dependent open → inactivated Quicker to open than K+ channels Voltage gated Na+ channel structure Voltage gated Na+ channel inactivation BIOL2174: Cell Physiology – Lecture Notes Voltage gated K+ channel Can exist in 2 states o Closed o Open Voltage gated o Voltage moves channel between o Closed → open o Open → closed Delayed activation o Opening takes ~5ms Slower to open compared to Na+ channels Action potential summary BIOL2174: Cell Physiology – Lecture Notes Lecture 6 – Action Potential Propagation Resistance Resistance Unit Ohm (Ω) Thin – larger resistance to charge movement (electrons) o Small conductance Thick – smaller resistance to charge movement o Large conductance Conductance = 1/resistance = σ o Conductance Unit = 1/ Ω or Siemens Membrane resistance Depends on o Resistance of open channels o Number of channels present in the membrane Membrane Resistance Unit = Ω/mm2 The more easily ions can cross the membrane, the lower its resistance o Less open channels = lower resistance Axial resistance How easily charge can flow along the cell Depends on o Diameter ▪ Wide = lower resistance o Number of charge carriers ▪ Saltier solution = lower resistance Capacitance C = q/V o C = capacitance o q = charge o V = voltage Membrane capacitance Membrane can act as a capacitor o Charges accumulate on each side V = q/C Capacitance is inversely proportional to membrane width o A wide membrane has a small capacitance and generates a large change in membrane potential for a given movement of ions BIOL2174: Cell Physiology – Lecture Notes Propagation of the action potential – Nerve conductance How does an action potential propagate along a cell: The ions move along the cell by diffusion from high concentration to low concentration and the Na+ channels inactivate behind the ions as the cells move along Why does the action potential only move in one direction? (Directionality) Due to the inactivation of Na+ channels behind impulse Due to the open K+ channels behind impulse What determines the frequency at which a nerve cell can send action potentials? (Conduction velocity) Myelin sheath surrounding the axon The width of the neuron Directionality 1: Some K+ channels remain open BIOL2174: Cell Physiology – Lecture Notes Directionality 2: Recovery time of Na+ channels after inactivation Pulse 1 opens than inactivates Na+ channels Recovery of inactivation is described by an exponential function – time o Constant (T) = 5ms Channels need to rest before they can reopen. Factors influencing the speed of propagation To increase velocity o Make it easier for ions to move along the axon 1. Make neuron wider – decrease the resistance of the pathway down the axon 2. Stop ion leakage out of neuron – increase membrane resistance o Make ions moving into the axon have a bigger effect 3. Increase membrane thickness – decrease membrane capacitance 1. Invertebrates decrease resistance by increasing diameter Decrease longitudinal resistance by increasing axon diameter Fatter – fibre – larger cross-sectional area Less longitudinal resistance, as current has more parallel pathways down the interior of the axon 2. Increase membrane resistance Some leakage of ions through membrane and open channels Preventing this, forces more ions along the axon 3. increase membrane thickness – decrease membrane capacitance Moving a single charge across a thick membrane has a larger effect on the membrane potential than moving the same charge across a thin membrane Moving the same number of ions more efficiently depolarises a thick membrane Increasing thickness (myelin) decreases capacitance o Thus, less ions have to move to change the membrane potential o Thus, depolarisation will spread faster BIOL2174: Cell Physiology – Lecture Notes Vertebrates Use myelin sheath o Myelin = non-neuronal glial cells o Increases membrane resistance o Increases membrane thick – decrease capacitance o Myelin slows conduction in small fibre o Myelin speeds conduction in large fibre Saltatory conduction BIOL2174: Cell Physiology – Lecture Notes Lecture 7 – Neuron Anatomy & Ion channel diversity and disease Cell body (Soma) Dendrites – receive input signals Axon hillock – where the axon joins to the soma – where the axon potential starts o Has a high diversity of ion channels Axon o Myelin sheath – Schwann cells – helps speed up the axon potential o Nodes of Ranvier – gaps in the myelin sheath – ion channels are clustered here (low diversity of ion channels) Axon termini – connects axon to other nerve cells Ion channel diversity Mutation In voltage-gated Na channels Faulty inactivation If a normal AP looks like this: BIOL2174: Cell Physiology – Lecture Notes Which of the following is the most likely shape of the AP in a cell which has faulty Na channels that are less likely or slower to inactivate – Answer = B due to slower repolarisation Mutations in neuronal voltage-gated Na channels Mammalian voltage-gated Na channel subtypes (alpha subunit) Case study – Developmental and epileptic encephalopathy (DEE) Epilepsy – Seizures Encephalopathy – significant developmental delay BIOL2174: Cell Physiology – Lecture Notes Blue is a normal Nav1.2 (wildtype) Purple is the mutant – only a little bit of inactivation – Na channels keep continuing Na+ Channel (in)activation curve At rest (~-70mV) channels will be neither inactivated or open At large +ve potentials the channels are likely to open, but are also likely to inactivate Common effect of disease-causing mutations is to move these curves left or right BIOL2174: Cell Physiology – Lecture Notes K+ channel diversity More than 200 K channels Why so many types of Na and K channels Differences in AP shape and frequency – shape used to encode information BIOL2174: Cell Physiology – Lecture Notes Channels can differ in: o Localisation o When they open (threshold) o Hold quickly they open o How quickly they inactivate o How quickly they recover K+ channel diversity Families of ion channels Roles of ion channels Sensing (sensory transduction) BIOL2174: Cell Physiology – Lecture Notes o External signals – touch, hearing, taste, pain o Internal – cell-cell communication, cell volumes, pressures, presence of nearby cells Conveying a message o Sending an AP o Regulating when and how often and how to combine APs Converting a message into a cellular response o Neurotransmitter release o Secretion of hormones o Contraction of muscle Families of ion channels ATP sensitive K channels and insulin secretion Non-electrical reason for channels BIOL2174: Cell Physiology – Lecture Notes Families of ion channels Families of ion channels Voltage gated channel superfamilies Ligand gated ion channels Mechanically gated channels Other channels o Chloride channels o Aquaporins (water channels) o Connexins o Sodium leak channels o Orai/CRAC channels (calcium release-activated calcium channels) Summary There are many kinds of Na, K and other channels all with slightly different biophysical properties The diversity of channels allows for fine control over membrane potentials and AP firing Channels also play important roles in other processes such as synaptic communications, sensory transduction, volume regulation, chemical regulation BIOL2174: Cell Physiology – Lecture Notes Lecture 8 – Channel Diversity and Drugs Differences of ion channels Differences in ion selectivity Activated by diverse stimuli Different sizes/shapes Mutations lead to channelopathies What is a drug A chemical substance that acts on a molecular target in our body to modulate its activity and have a therapeutic effect The drug development process Voltage-gated Na channels Commonly used Na channel drugs Anti-seizure Anti-arrhythmic Anti-paralytic BIOL2174: Cell Physiology – Lecture Notes Local anaesthetic Routes for Drug Binding Understanding Drug Interactions Using simulations, we investigate a. How Na channels behave in the cell b. How non-selective drugs interact with the Nav channel (how they pass through the fenestrations & where they bind in the pore) c. How to improve drug selectivity and reduce side effects BIOL2174: Cell Physiology – Lecture Notes Can we achieve subtype selective pore block via fenestration access? How do drugs bind in the pore Case 1 – the quest for non-opioid treatment for pain BIOL2174: Cell Physiology – Lecture Notes Case 2 – toxic side effects & the anti-target hERG 90% of drugs fail clinical trails The main reason is off-target toxicity hERG (Human ether-a-go-go) channel is responsible for the rapid delayed repolarising K+ current hERG inhibitions can lead to fatal arrhythmia Cisapride o Serotonin receptor agonist o Withdrawn from market in the 1990s due to increased risk of severe arrhythmias o Inhibits hERG channels o Use is now restricted – mostly for veterinary applications Drugs inspired by nature Digoxin – derived from Foxgloves o Inhibits the Na+/K+ pump o Treats atrial arrhythmias o Narrow therapeutic change Cannabidol – derived from Marijuana plant o Recently approved for treatment of seizures o 65 targets, including cannabinoid receptors, serotonin receptors, Nav channel, TRPVI channel Semaglutide – discovered from Gila monster venom BIOL2174: Cell Physiology – Lecture Notes o Peptide drug o Activates the GLP-I receptors o Increases insulin secretion o Treats type 2 diabetes Small molecule vs small peptide drugs Small molecule o Most commercial drugs are small molecule compounds o Chemically synthesised o Small molecule weight - ~100-500 Daltons o High membrane permeability & oral bioavailability (pro) o High metabolic stability (pro) o Poor selectivity (con) o More toxic side effects (con) Small peptide drugs o 2-100 amino acid sequences o Earliest use case – insulin in the 1920s o Highly selective for a target (pro) o Less toxic side effects (pro) o Poor membrane permeability & oral bioavailability (con) o Short half-life (con) Case 3 – Killer cone snails help fight pain Selectively inhibits mammalian N-type/Cav2.2 channels that are responsible for neurotransmitter release into the DRG neurons involved in pain sensation Ziconotide is a peptide drug approved in 2004 for treating intractable pain Drug needs to be administered intrathecally (spinal fluid) Drugs & toxins can bind different regions of Nav channels 1. Central pore cavity o Small molecule Nav drugs o Batrachotoxin (poison dart frog) 2. Extracellular pore opening o u-conotoxin (cone snail) o Tetrodotoxin (puffer fish) 3. Voltage sensors o Gating modifier toxins (spider, scorpion, ant, plants) o Novel small molecule developments BIOL2174: Cell Physiology – Lecture Notes Case 4 – targeting Nav voltage sensors Summary What is a drug The drug development process is expensive and has a high failure rate due to various challenges Many drugs work by targeting ion channels There are many types and subtypes of ion channels Selective targeting of one type of channel can lead to reduced toxicity/side effects and greater therapeutic efficacy Differences between small molecule and peptide drugs Investigating natural products can lead to better understanding of how to target ion channels and potential discovery of novel drugs Targeting different regions of an ion channel can lead to different effects BIOL2174: Cell Physiology – Lecture Notes Lecture 9 Sensory transduction (pre lecture video) The process of converting a sensory signal to an electrical signal in a sensory neuron that can create a response or be interpreted by the brain Opening a nonselective, cation selective or Na selective channel will cause Na+ to enter the cell → increase the membrane potential → initiate an AP End point is always opening or closing an ion channel → creates an electrical signal for the AP to be interpreted by the brain Specialised neurons or specialised cells connecting to neurons → detect sensory stimulus Sensory transduction The conversion of a stimulus into an electrical signal, via an ion channel Sight – the molecular basis BIOL2174: Cell Physiology – Lecture Notes Opsin activation → GPCR signally cascade → AP in optic nerve Sight – what happens when things go wrong? Mutations in rhodopsin gene can lead to vision loss (the grey one in the image above) Mutations in cone opsin genes lead to colour blindness o Red-green colour blindness is caused by loss or mutation of the OPN1LW or OPN1MW genes o Sex-linked condition – 8% of males, 0.5% of females Hearing – what is the molecular basis Mechanically gated ion channel is activated by pulling motion of the inner hair cells Hearing – what happens when things go wrong? Beethoven mice – missense mutation in TMC1 dn mice – missing TMC1 Gene therapy can restore hearing Mechanically gated ion channels Found in all kingdoms of life Structural diversity indicates independent evolution Ability to sense physical forces essential for survival BIOL2174: Cell Physiology – Lecture Notes Piezo channels Piezo 1 o Important for blood circulation, bone development, heart function Piezo 2 o Sense touch, proprioception and interoception Unique shape causes membrane dome Membrane proteins and lipids Lipids play important roles in the regulation of ion channels, especially mechanically gated ion channels BIOL2174: Cell Physiology – Lecture Notes Touch – what is the molecular basis Mechanoreceptors o Skin stretch/light touch o Texture/vibration o Deep pressure o Sustained touch/pressure Thermoreceptors o Warmth receptors o Cold receptors Pain receptors (nociceptors) Proprioceptors Mechanically gated ion channels TRP channels Pain – what is the molecular basis Pain is a response to noxious stimuli What external stimuli causes pain? o Mechanical, thermal, chemical Smell – what is the molecular basis BIOL2174: Cell Physiology – Lecture Notes Taste – what is the molecular basis Summary The conversion of a stimulus into an electrical signal, via an ion channel Disruptions to this pathway can lead to sensory disorder Mechanically gated ion channels help us sense physical forces BIOL2174: Cell Physiology – Lecture Notes Lecture 10 – Synapses Synaptic transmission Synapse – connection between neurons or between neuron and muscle cell Brain has 100 billion neurons Each axon has about 1000 branches connecting to other neurons o Meaning 100 trillion synapses Electrical Synapse Less common Direct current flow from one cell to another through gap junctions channels (connexins) Allow bidirectional transfer Fast Allow synchronicity BIOL2174: Cell Physiology – Lecture Notes Chemical synapse Most synapses Rely on neurotransmitter release and receptor activation Postsynaptic receptor can gate an ion channel directly or indirectly Speed of electrical and chemical synapses Synapse anatomy BIOL2174: Cell Physiology – Lecture Notes How many different kinds of proteins are required for the chemical synapse to function Ion Channels o Nav o Kv o Cav o NaK pump Acetylcholesterase (enzyme) Cytoskeleton protein Fusion proteins (2 kinds) 2 o Get ready o Ca2+ dependent AchR ligand gated ion channel Vesicle formation proteins Neurotransmitter transporters Excitatory synapse Excitatory receptors depolarise (increase in membrane potential) the postsynaptic cell Makes it easier to start an action potential in the postsynaptic cell Excitatory neurotransmitters Acetylcholine (ACh) o Main excitatory NT at the neuromuscular junction with skeletal muscles Amino acid neurotransmitters o Main excitatory NT at nerve – nerve synapse BIOL2174: Cell Physiology – Lecture Notes Inhibitory synapse Ligand gated channels are anion channels instead of cation channel Inhibitory receptors hyperpolarise (decrease membrane potential) the postsynaptic cell Makes it harder to start an action potential in the postsynaptic cell; Inhibitory neurotransmitters GABA Glycine Summary The connections between nerve cells or nerve and muscle cells are known as synapses, of which there are 2 kinds electrical and chemical Chemical synapses make use of neurotransmitter and receptors to carry a signal from one cell to another Excitatory synapses involve ligand gated cation channels, that open upon the binding of NTs such as glutamate or acetylcholine to raise the membrane potential in the postsynaptic cell BIOL2174: Cell Physiology – Lecture Notes Inhibitory synapses involved ligan gated anion channels, that open upon the binding of NTs such as glycine or GABA to lower the membrane potential in the postsynaptic cell Why have both AMPA and NMDA receptors BIOL2174: Cell Physiology – Lecture Notes Lecture 11 – Synaptic integration & plasticity Synaptic integration (or summation) Combining multiple synaptic signals The size of EPSP depends on synapse position Spatial summation – multiple inputs can combine to reach threshold Temporal summation – multiple firing of single input can reach the threshold In general, a single EPSP is not enough to stimulate an AP (but not always) Strength of signal decreases with distance from cell body Thus, multiple signals are often combined An AP will only fire if the sum of EPSPs and IPSPs reaches the threshold voltage BIOL2174: Cell Physiology – Lecture Notes How many inputs are there on a given nerve or muscle One muscles fibre is innervated by a single motor neuron But a single motor neuron can innervate many muscle fibres Usually, mani presynaptic neurons synapse to every postsynaptic neuron Synapses can form, break and be strengthened or weakened. Summary 1 Multiple synaptic inputs can be combined using the concepts of spatial and temporal summation Only if the resultant effect reaches threshold at the axon hillock will an AP be initiated How can you strengthen or weaken the synapse Changing the probability that an AP firing in the pre-synaptic cell will cause an AP in the post- synaptic Avenues for plasticity Short-term plasticity – ms to second (presynaptic side) o Alter the ability of Ca2+ to enter o Alter the amount of NT released o Short term depression → vesicle depletion & Ca2+ channel inactivation o Short term facilitation → enhanced vesicle exocytosis & residual Ca2+ or lasting influence of Ca2+ o a-latrotoxin (from black widow spider) → forms Ca2+ permeable channels ▪ Massive NT release ▪ NT depletion ▪ Inability to fire synapse = flaccid paralysis Long term plasticity – up to years (postsynaptic side) – e.g., strengthening connections for memory o Alter the no. of receptors o Alter the permeability o Prolonged strong stimulation → Influx of Ca2+ leads to insertion of more AMPAR into membrane (long term potentiation) ▪ Phosphorylation of AMPAR → increase conductance BIOL2174: Cell Physiology – Lecture Notes o Prolonged weak stimulation → increase of Ca2+ leads to removal of AMPAR from membrane (long-term depression) o Spatial/temporal profile of Ca2+ must be important Neurotransmitters Wiring NTs o Ionotropic & metabotropic ▪ Acetylcholine & glutamate ▪ GABA & glycine Modulatory NTs (monoamines) o Mostly metabotropic o Capable of ‘volume transmission’ o More complex effects ▪ Serotonin (5-HT) ▪ Histamine ▪ Dopamine ▪ Epinephrine ▪ Norepinephrine Volume transmission BIOL2174: Cell Physiology – Lecture Notes Neuromodulation – the physiological process by which a given neuron uses one or more chemicals to regulate diverse populations of neurons o Neuromodulators typically bind to metabotropic G-protein couple receptors (GPCRs) to initiate a 2nd messenger signally cascade that induces a broad, long-lasting signal o This modulation can last for hundreds of ms to several minutes o Some of the effects include alter intrinsic firing activity, increase or decrease voltage- dependent currents, alter synaptic efficiently, increase bursting activity and reconfiguration of synaptic connectivity Summary 2 Synapses can be strengthened or wakened due to the pattern of use by altering the number of channels on the postsynaptic cell Synapse can be modulated by nearby neurons BIOL2174: Cell Physiology – Lecture Notes Lecture 12 – Muscle: Excitation-Contraction Coupling Pre-lecture video: Ca2+ channels Ca2+ transporters Voltage gated calcium channels Convert electrical impulse into action BIOL2174: Cell Physiology – Lecture Notes Family tree of voltage gated calcium channels L Ligand gated calcium channels Ryanodine receptors (RyR) 1,4,5-triphosphate receptors (IP3R) Ca2+ Neurotransmitters release More Ca2+ increases Ach release which creates large change in postsynaptic potential (= end plate potential EPP) Mg inhibits Ach release by competing with Ca2+ Ca2+ channels Cytosolic Ca2+ concentration is very low Voltage gated Ca2+ channels play the key role of converting electrical impulses into cellular responses Muscles Skeletal muscle structure BIOL2174: Cell Physiology – Lecture Notes Skeletal muscle fibre structure A whole new set of names for muscles o Sarcolemma = outer cell (plasma) membrane o Sarcoplasm = cytoplasm o Sarcoplasmic reticulum (SR) = ER ▪ Internal Ca2+ stores o Excitatory postsynaptic potential ▪ End plate potential o Transverse tubule (T-tubule) – invaginations into muscle cell Myofibril Patterns comes from interwoven thick (myosin) and thin (actin) filaments along with joining proteins Contractile apparatus Troponins hold tropomyosin in position to block myosin binding site Binding of Ca2+ to troponin uncovers myosin binding site BIOL2174: Cell Physiology – Lecture Notes Muscle contraction – cross bridge cycle BIOL2174: Cell Physiology – Lecture Notes If you run out of ATP you will strop the process and the muscle will be contracted Removing Ca results in muscle relaxation Skeletal excitation-contraction coupling DHPR-voltage gated calcium Channel (L-type Cav1.x) RyR1 – ligand gated calcium Channel directly gated by DHPR Cardiac Excitation-contraction coupling DHPR-voltage gated calcium Channel (L-type Cav1.x) RyR2 – ligand gated calcium Channel gated by Ca2+ BIOL2174: Cell Physiology – Lecture Notes Malignant Hyperthermia After exposure to general anaesthetic such as halothane, muscles become stiff and body temperature rises greatly Problem is caused by interaction of anaesthetic with, or faulty SERCA Ca2+ pump, and in this case the Myosin ATP motor BIOL2174: Cell Physiology – Lecture Notes Lecture 13 – Uniporters Transport proteins act in concert The net uptake (influx) into, and extrusion (efflux) from cells, (as well as into and out of subcellular organelles) is achieved by a combination of; o Primary active transport (pumps) o Secondary active transport (symporters & antiporters) o Passive transport (uniporters & channels) They all act in concert with one another Primary active transport (pumps) E.g., uptake of glucose in the intestine BIOL2174: Cell Physiology – Lecture Notes E.g., uptake of glutamate from the synapse BIOL2174: Cell Physiology – Lecture Notes Uniporters The GLUT (SLC2A) family of transporters (uniporter) GLUT = Glucose Transporter o Integral membrane proteins with 12 membrane-spanning helices o Transport glucose and related hexoses via a ping-pong (alternating confirmation) mechanism o There are 14 members of the GLUT family expressed in humans o Many play a specific role in glucose metabolism determined by their pattern of tissue expression, substrate specificity, transport kinetics, and regulated expression in different physiological conditions o The primary substrate for at least some of the GLUT transporters is unknown o Members of the family play a role in glucose uptake in other organisms Ping pong mechanism (alternating confirmation) Inward and outward facing confirmations Trans-stimulation Radiolabelled glucose effluxing from a red blood cell BIOL2174: Cell Physiology – Lecture Notes How does unlabelled glucose outside the cell speed up (i.e., trans-stimulate) the efflux of labelled glucose from inside the cell? o The confirmational change is faster when there is a glucose molecule → the rate at which glucose is coming out of the cell is limited by how fast the confirmational change can happen, the confirmational change happens faster when there is a substrate bound than when there isn’t → trans-stimulation Kinetics GLUT1 (aka SLC2A1) Expressed at high levels in erythrocytes (red blood cells) and in the endothelial cells of barrier tissues such as the blood-brain barrier Responsible for the low level of basal glucose uptake required to sustain metabolism in many cell types Mutations in the GLUT1 gene are responsible for the Glucose Transporter type 1 deficiency syndrome (GLUT1 DS), also known as De Vivo disease BIOL2174: Cell Physiology – Lecture Notes GLUT1 DS is characterised by a low cerebrospinal glucose concentration which results from impaired glucose transport across the blood-brain barrier Children with this disease present with seizures, ataxia (gross incoordination of muscle movements), microcephaly, developmental delay Treatment → a ketogenic diet provides ketones as an alternative fuel to the brain, instead of glucose. GLUT2 Expressed in kidney (renal tubular) cells and intestinal cells, as well as in the liver and in pancreatic beta cells GLUT3 Expressed in neurons and in the placenta GLUT4 Expressed in adipose tissue and striated muscle (skeletal muscle & cardiac muscle) Regulated by insulin Insulin recruits GLUT4 to the plasma membrane of adipose and muscle cells from intracellular storage sites BIOL2174: Cell Physiology – Lecture Notes Insulin-induced recruitment of GLUT4 GLUT4 and type 2 diabetes States of insulin resistance such as type 2 diabetes are associated with impaired regulation of GLUT4 gene expression and function GLUT1 in cancer cells Reprogramming of cellular energy metabolism is a hallmark of cancer cells Most cancer cells use glycolysis (fuelled by glucose) as their main ATP production method instead of mitochondrial oxidative phosphorylation GLUT1 (SLC2A1) is overexpressed in many types of cancers, including the brain, colon, kidney, lung, ovary, prostate Overexpression of GLUT1 (SLC2A1) has been associated with poor prognosis in some cancer- types Preclinical studies have demonstrated that inhibiting GLUT1 can lead to diminished tumour growth in vitro and in vivo GLUT1 inhibitors, when administered in combination with other chemotherapeutic agents, show synergistic antitumour effects The 14 GLUT transporters in humans share high sequence homology – many GLUT inhibitors lack isoform specificity The development of GLUT1-specific inhibitors as potential anti-cancer agents may benefit from the understanding of the atomic structure of GLUT1 and the identification of key regions of the protein that could be targeted for selective inhibition BIOL2174: Cell Physiology – Lecture Notes GLUT homologues in other organisms – malaria parasite Malaria parasite has a homologue of the GLUT protein, called PfHT1 PfHT1 plays a key role in the uptake of glucose, an essential metabolic fuel for parasite PfHT1 is inhibited (competitively) by a compound referred to as compound 3361 PfHT1 Glucose uptake into the malaria parasite in the presence and absence of the glucose transporter inhibitor compound 3361 Effect of compound 3361 on ATP levels in the parasite Compound 3361 kills the parasite inside the red blood cell, so as inhibitor of the parasites glucose transporter may be an effective antimalarial BIOL2174: Cell Physiology – Lecture Notes Lecture 14 – Secondary active transport p.1 Secondary active transport → symporters & antiporters We have a lot of secondary active transporters (over 400) We have fewer primary active transporters Secondary active transport is typically what we use to move things against gradients Symporters Also called cotransporters Antiporters Also called exchangers BIOL2174: Cell Physiology – Lecture Notes Secondary active transport This system mediates the transport of substrates up the electrochemical gradient by coupling this process to the movement of a second substrate does its electrochemical gradient o Often, but not always Na+ The physiological functions of these systems include the active accumulation of nutrients, NTs and ions within cells, as well as the regulation of cytosolic [Ca2+], cytosolic pH and cell volume Gradients Solutes move down a concentration gradient o The movement of molecules down a concentration gradient is a consequence of the random diffusion of the molecules in solution o An individual molecule does not experience a force pushing it in a particular direction o Movement against a concentration gradient requires an input of energy o Movement down a concentration gradient liberates energy Solutes move down an electrochemical gradient o If the solute carries an electrical charge, and if there is a voltage across the membrane o E.g., below, the fan represents that membrane potential, acting on the charge carried by the diffusing species o An individual ion does experience an electrical force pushing it in a particular direction Electrochemical gradient In the term electrochemical gradient – the chemical refers to the concentration gradient, the electro refers to the electrical gradient Molecules or ions have a natural tendency to move down an electrochemical gradient To move molecules or ions up an electrochemical gradient require an input of energy BIOL2174: Cell Physiology – Lecture Notes Conversely, the movement of molecules or ion down an electrochemical gradient liberates energy Determines direction of ion movement Is a form of stored (potential) energy The amount of energy associated with the concentration (chemical) and electrical (electro) components of the gradient for a particular solute, S, is expressed as: These equations together are a quantitative expression of the electrochemical gradient for a solute, S Calculating the energy changes associated with molecules or ions moving up or down an electrochemical gradient o For N moles of a solute (S), moving across a member the energy change can be calculated from the expression: o For a solute moving down an electrochemical gradient ΔG0 Stoichiometry Example 1 – Cotransport with 1:1 stoichiometry (e.g., 1 Na+:1 glucose) o At electrochemical equilibrium, the amount of energy liberated by Na+ moving down its gradient will equal the amount of energy being used to move glucose against its gradient BIOL2174: Cell Physiology – Lecture Notes o So under these conditions a transporter with a 1:1 stoichiometry can move glucose into the cell and establish an intracellular concentration that is 95-fokd higher than the extracellular concentration Example 2 – cotransport with 2:1 stoichiometry (e.g., 2 Na+: 1 glucose) o At electrochemical equilibrium the amount of energy liberated by Na+ moving down its gradient will equal the amount of energy being used to move glucose against its gradient o I.e., the amount of energy required to move 1 glucose molecule against its concentration’s gradient is exactly balanced by the amount of energy liberated by 2Na+ ions moving down its electrochemical gradient o So under these conditions, a transporter with a 2:1 stoichiometry can move glucose into the cell and establish an intracellular concentration that is nearly 9000-fold higher than the extracellular concentration BIOL2174: Cell Physiology – Lecture Notes Manipulating both the [Na+] and Vm components of the electrochemical gradient in membrane vesicles Valinomycin is a K+ ionophore (changes the permeability of a particular ion), a chemical agent that increases PK Vesicle overshoot experiment (glucose uptake into apical membrane vesicles) When [Glucose]i/[Glucose]O = 1, the concentration of glucose inside the vesicle is the same as the concentration outside the vesicles o I.e., there is no concentration gradient When [Glucose]i/[Glucose]O > 1, there is an outward glucose concentration gradient o Remember that the generation of a concentration gradient always requires energy The Na+ concentration gradient is manipulated simply by manipulating the concentration of Na+ inside and outside the vesicles Vm is manipulated by manipulating the concentration of K+ inside and outside the vesicles and by adding the K+ ionophore valinomycin When there is no Na+ present, there is no glucose transport via the Na+:glucose transporter o The slow uptake you can see into the vesicles is leakage into the imperfectly-sealed vesicles Where there is Na+ present, but no [Na+] gradient and no voltage, the Na+:glucose transporter works but cannot build up a concentration gradient o It is effectively functioning as a uniporter, with Na+ bound at the active site The Na+:glucose transporter can build up a glucose concentration gradient when there is: 1. A Na+ concentration gradient, or 2. An electrical gradient (i.e., an inward negative membrane potential), or 3. When there is both a Na+ concentration gradient and an electrical gradient BIOL2174: Cell Physiology – Lecture Notes Lecture 15 – Secondary active transport, p.2 SGLT 1 Sodium glucose transporter 1 A relatively low Km (~0.4mM) and a relatively low Vmax SGLT2 Sodium glucose transporter 2 A relatively high Km (6 mM) and a relatively high Vmax SGLT1 and SGLT2 Both are present in the proximal tubule of the kidney, where they play a central role in the reabsorption of glucose from the renal filtrate (so that the urine contains as little glucose as possible) Kidney function Formation of urine by the kidney involves 3 processes 1. Ultrafiltration of plasma by the glomerulus 2. Reabsorption of water and solutes from the ultrafiltrate 3. Secretion of selected solutes into the renal tubular fluid The 2nd of these occurs along the length of the proximal tubule The proximal tubule reabsorbs approx. 67% of the filtered water, Na+, Cl- and K+ o In addition, virtually all of the glucose and other nutrients (e.g., amino acids) filtered by the glomerulus are reabsorbed BIOL2174: Cell Physiology – Lecture Notes Most of the reabsorption of nutrients such as glucose occurs along the first third of the proximal tubule o More distal segments reabsorb almost all of the remainder In the initial filtrate produced at the glomerulus the concentration of glucose is the same as that in the blood plasma As the filtrate moves down the proximal tubule and the glucose is reabsorbed, the concentration in the filtrate is reduced, hence the need for active transport Thermodynamics vs kinetics The stoichiometry (i.e., the no. of Na+ ions being transported per sugar molecule) determines the amount of energy released each time the transporter goes through the transport process o This determines the size of the sugar concentration gradient that this transporter can In theory establish o Or, put another way, the max size of the gradient against which this transport can in theory transport sugar molecules In considering the energetic implications of transporter stoichiometry ew invoked the concept of Gibbs free energy (G) → this is a thermodynamic concept o The change in G (ΔG) associated with any particular process (whether is be the transport of something across a membrane or a chemical reaction) will tell you whether the process can occur spontaneously Any process which causes the G to decrease (i.e., ΔG 200 Menkes disease causing mutations in ATP7A have been identified Symptoms include neurological defects, slow growth, hypothermia, steely hair Sever forms usually fatal before the age of 3 yrs Mild forms can be treated with a naturally occurring copper amino acid complex – copper- histidine Incidence ~ 1:100,000 and 1:250,000 X-linked – more common in males Wilson’s disease Mutation of ATP7B ATP7A Shares homology with ATP7A Most abundantly expressed in the liver, also found in brain and other sites BIOL2174: Cell Physiology – Lecture Notes A major role is to excrete Cu+ into the bile Travels between the Golgi and the cell periphery (and bile canaliculus in the case of hepatocytes) Mutations can lead to impaired Cu+ secretion into the bile Wilson’s disease Caused by mutations in ATP7B that result in copper overload Copper accumulates primarily in the liver and brain, causes tissue damage ~ 300 mutations in ATP7B identified – some occur commonly Symptoms include liver cirrhosis, hepatitis, vomiting, weakness, swelling of legs, yellowish skin, tremors, anxiety, a ring around the cornea made form deposited copper Treated w/ dietary changes including a low-copper diet (no chocolate), medications to decrease copper absorption or remove copper form body Incidence ~ 1:30,000 With early detection and treatment, can often live a relatively normal life Autosomal recessive P-type ATPases – domain architecture TM (or M) (transmembrane) domain o Usually contains 10 TM helices o Substrate binding and translocation 3 cytosolic domains o P – phosphorylation ▪ Contains a highly conserved signature sequence → DKTGT ▪ The D (aspartate) is phosphorylated during the transport cycle o A – actuator ▪ Contains a conserved TGE motif that interacts with the phosphorylation site o N – nucleotide-binding ▪ Binds ATP BIOL2174: Cell Physiology – Lecture Notes P-type ATPases – general transport mechanism Post Albers cycle 1. Substrate (X+) & ATP binding 2. ATP hydrolysis → phosphorylation 3. Binding site reorientation 4. Substrate dissociation 5. Dephosphorylation (aided by A-domain) 6. Reorientation (resetting) Some p-type ATPases require interaction with a beta-subunit (e.g., the Na+/K ATPase) Operational model of the PM Na+/K+ ATPase This pump is a drug target – people inhibit it on purpose BIOL2174: Cell Physiology – Lecture Notes The mechanism of action of cardiac glycosides Cardiac glycosides E.g., Ouabain, digoxin o Used for centuries as medicines and poisons o 18th century – foxglove extracts used to treat edema o 1799 – heart identified as primary site of action of foxglove o 19th century – digitalis (plant extract containing digoxin) used indiscriminately for treatment of cardiac problems, often with fatal outcomes o By early 20th century – digoxin used more judiciously for congestive heart failure o 1983 – digoxin is the 4th most commonly prescribed drug in the US BIOL2174: Cell Physiology – Lecture Notes Lecture 20 – Primary active transport 2 Cardiac glycosides – mechanism of action They inhibit the Na+/K+ pump, particularly the heart isoform Ca2+ release from sarcoplasmic reticulum is important for strong heartbeat Partially inhibiting the Na+/K+ pump helps more Ca2+ get into the SR After each heartbeat the cytosolic Ca2+ has to be brought back down to its very low resting concentration The cell has multiple mechanisms to do this 1. The SERCA (a P-type ATPase) pumps Ca2+ into the SR 2. The PMCA (a p-type ATPase) pumps Ca2+ out of the cell 3. The Na+/Ca2+ exchanger (NCX) uses the energy in the inward Na+ electrochemical gradient to transport Ca2+ out of the cell IN people w/ congestive heart failure, the sarcoplasmic reticulum can be more leaky to Ca2+ Partial inhibition of the heart isoform of the Na+/K+ pump reduces the inward Na+ electrochemical gradient across the PM of the heart cell This means that NCX transports less Ca2+ out of the cell, and more of the Ca2+ gets pumped back into to the SR This means that there is more Ca2+ in the SR to be released for the next heartbeat P4-ATPases Membrane asymmetry A feature of the PM and also certain organelle membranes (e.g., Golgi, endosomal membranes) PS and PE are typically more abundant in the inner/cytoplasmic leaflet, whereas SM and PC are more abundant in the outer leaflet Flip → go from outer leaflet to inner/cytoplasmic leaflet Flop → go form inner/cytoplasmic leaflet to outer leaflet BIOL2174: Cell Physiology – Lecture Notes Membrane asymmetry is important for many reasons, including membrane trafficking processes and certain signally events PS in the outer leaflet serves as an eat me signal to macrophages Proteins involved in membrane asymmetry 1. P4-ATPases (flippases) → flip phospholipids (usually PS and/or PE) from the outer leaflet to the inner leaflet – ATP-dependent 2. Floppases (certain ABC transporters) → flop phospholipids – ATP-dependent 3. Scramblases → mediate random bidirectional movement of lipids – not ATP-dependent (but dependent of Ca2+ or activation by caspases) P4-ATPases – role and distribution AKA type IV P-type ATPases or phospholipid flippases Role – to flip phospholipids (usually PS and/or PE) from the outer leaflet to the inner leaflet P4-ATPases are found across eukaryotes Humans have 14 P4-ATPases BIOL2174: Cell Physiology – Lecture Notes P4-ATPases – structure Like other P-type ATPases they: o Hydrolyse ATP o Are sensitive to vanadate o Contain M, N, P, A domains o Contain DKTGT motif Most P4-ATPases need a beta-subunit (from the CDC50 family of proteins) P4-ATPases – mechanism Mechanism of transport is similar to other P-type ATPases (Post Albers Cycle) Only one substrate transported (and only in one direction) The E1 phases (ATP binding, phosphorylation of the P domain) is still important for preparing for transport but is transport-salient The phospholipid flip is coupled with the dephosphorylation of the conserved aspartate The headgroup of the phospholipid goes through the transport pathway until it reaches the right place in the inner leaflet BIOL2174: Cell Physiology – Lecture Notes How to study P4-ATPAses Measure ATP hydrolysis Use fluorescently labelled lipids, e.g., NBD-PS o Allows cells to internalised NBD- phospholipid o To stop internalisation at different time points – scavenged NBD-phospholipid that is extracellular or in the outer leaflet with BSA (bovine serum albumin) o Measure the fluorescence that has been incorporated The V-type ATPase Overview Named for its identification on the membranes of Vacuoles Highly conserved in all domains of life – archaea, bacteria, eukaryotes Responsible for pumping H+ into acidic organelles Also found on the plasma membrane in some cell types An amazing piece of machinery Inhibited by concanamycin V-type H+ ATPase – domain architecture V1 domain o ATOP hydrolytic domain o ~ 650 kDa o Cytosolic side of membrane o Composed of 8 different subunits → A (x3), B (x3), C, D, E (x2), F, G (x2), H (1 or 2) V0 domain o H+ translocation domain o ~ 260 kDa o Embedded in the membrane o Composed of 6 different subunits → a, d, e, c (x4-8), c’ (or in some organelles Ac45), c” o The proteolipid subunits c, c’, and c” are very hydrophobic and for a ring Central stalk o Comprises the D and F subunits of V1 and the d subunit of V0 Peripheral stalks o Consists of the C, E, G, H subunits of V1, and N-terminal domain of subunit a (V0) In mammals, multiple isoforms exists for some of the subunits BIOL2174: Cell Physiology – Lecture Notes V-type H+ ATPase – structure The protein receptor interacts with the V-type H+ ATPases as an accessory protein – link between the V-type H+ ATPases and signalling pathways including the renin-angiotensin system for the regulation of blood pressure and electrolyte balance V-type H+ ATPase – transport mechanism Rotary mechanism 3 catalytic sites for ATP hydrolysis – A/B interface Central stalk rotates, coupling the energy released from hydrolysis of ATP to the rotation of the ring in V0 Peripheral stalks stop the A3B3 head from rotating Subunit possesses 2 hemi-channels and an important arginine residue (R735) – required for H+ translocation The proteolipid c, c’, c” subunits each have a buried Glu residue that undergo reversible protonation during H+ transport BIOL2174: Cell Physiology – Lecture Notes ATP hydrolysis between the A/B subunits drives rotation of the central rotor The central stalk (D, F, d) and the c, c’, c” ring rotate Rotation of the proteolipid ring relative to subunit a result in H+ transport Subunit a provides paths (aqueous hemi-channels) for H+ entry and exit to the protonatable Glu residues of the c, c’, c” subunits Deprotonation induced by interaction with Arg residue in subunit a 3 catalytic ATP-binding sites in V1 → 6-10 protonable sites on the proteolipid ring in V0, predicted (and measured) stoichiometry is 2-3.3 H+ translocated/ATP hydrolysed BIOL2174: Cell Physiology – Lecture Notes Lecture 21 – Primary Active Transport 3 V-Type ATPase Roles of the V-type H+ ATPase in our cells Generation of H+ gradient to drive NT uptake into vesicles Important role in the acidification of intracellular organelles Also present on the plasma membrane in some of our cells Osteoclasts express the V-type H+ ATPase on the plasma membrane Some cancer cells express the V-type H+ ATPases on the plasma membrane The V-type H+ ATPase – Regulation Post-translational modifications – phosphorylation, glycosylation Reversible dissociation of the V1 and V0 domains o E.g., dissociation happens in yeast when they are depleted of glucose – details of how this happens is not fully determined; prevents both H+ movement and ATP hydrolysis; conserves energy Control of localisation o E.g., in the case of PM localised pumps – reversible exocytosis and endocytosis in areas with a high density of pumps Interacting proteins involved in each case ABC transporters Overview ABC transporters are ancient and present in species of all kingdoms Human ABC transporters – some are multidrug efflux systems, some are not ATP-Binding Cassette (ABC) Diverse substrates Bacteria have ABC transporters involved in nutrient import as well as ABC exporters Most human ABC transporters are thought to be exporters – a variety of natural and synthetic substrates Well known for their roles in mediating resistance to anticancer drugs BIOL2174: Cell Physiology – Lecture Notes ABC transporters – domain architecture Typically consists of 2 nucleotide-binding domains (NBDs) and 2 transmembrane domains (TMDs) The domains are not always on the same polypeptide – some are formed y the dimerisation of 2 half-transporters each containing 1 NBD and 1 TMD NBDs o Bind and hydrolyse ATP o Cytosolic o Contain conserved sequence motifs ▪ LSGG(Q/R/K)QR = signature sequence ▪ GXXGXGKSS/T; hhhhD – Walker A/B = ATP binding (h is a hydrophobic residue) o Form dimers, which can open and close TMDs o Substrate binding site(s) o Translocation conduit o No conserved sequence – varied substrates ABC transporters – transport mechanism Some diversity in transport mechanisms Things that need to happen to transport a substrate (not necessarily in this order) o Substrate binding o Binding of 2 ATP molecules to the NBDs o NBD dimerisation o Confirmational change – inward to outward facing o ATP hydrolysis o Substrate release o Release of ADP and Pi o Resetting ABC transporters – natural functions and roles in disease Many ABC transporters play important roles in our cells, with mutations in them causing disease ABC transporters including ABCB11, ABCB4, ABCG5-ABCG8 are involved in bile production o Dysfunction association with liver disease ABCD1 transports very long chain fatty acyl-CoAs (VLCFA-CoAs) into peroxisomes BIOL2174: Cell Physiology – Lecture Notes o Dysfunction associated w/ X-linked adrenoleukodystrophy ABCD1 – natural function Fatty acids are important for numerous biological processes, including the creation of biological membranes Fatty acids are ingested, synthesised by the body, elongated, broken down – delicated balance to regulate abundance Role of ABCD1 is to transport very long chain fatty acyl-CoAs (VLCFA-CoAs; > C20) into peroxisomes VLCFA-CoAs are broken down in the peroxisomes in the process of b-oxidation ABCD1 and X-linked adrenoleukodystrophy Mutations in ABCD1 give rise to X-ALD, a disease in which this process does not occur effectively, leading to a build-up of VLCFAs 600 different mutations have been identified – clinical heterogeneity Incidence - ~1:20,000 males The accumulation of VLCFAs occurs over years, results in demyelination – disease typically becomes evident in childhood The disease often results in sever neurodegenerative decline Female carriers typically avoid severe disease, but can show symptoms later in life Lorenzo’s oil o Consists of shorter chain fatty acids – erucic acid (C22 monounsaturated) oleic acid (C18 monounsaturated) – aim to displace and reduce the synthesis of the longer chain fatty acids o Formulated by Lorenzo Odone’s parents o Normalises the levels of VLCFAs in the blood o Unfortunately does not reverse damage done to the brain – may have utility when treatment starts early o Lorenzo died at the age of 30 – much later than initially predicted ABCD1 Belongs to the D family within the ABC transporter superfamily A half transporter that forms dimers Studying purified (mostly human) ABCD1 using ATPase assays: BIOL2174: Cell Physiology – Lecture Notes ABCD1 structures ABC transporters – roles in drug resistance BIOL2174: Cell Physiology – Lecture Notes Lecture 22 – Primary Active Transport 4 Classes of primary active transporters Multidrug efflux systems involved in resistance to anticancer drugs P-glycoprotein (aka ABCB1) Multiple isoforms of Multidrug Resistance-associated Proteins (MRPs; aka ABCCs) Breast Cancer Resistance Protein (BCRP, aka ABCG2) Theses transporters all hydrolyse ATP and pump diverse drugs out of cancer cells – they are major obstacles to effective treatment Substrate specificities – some overlap, some differences Recent explosion in cryo-EM structure ABCCs in resistant cancers ABCC1 → identified in 1992 from drug-resistant cancer cells that did not express ABCB1 o Role ▪ Role in drug resistance established in numerous cancers (multiple types of leukemia, non-small-cell lung cancer, prostate cancer, breast cancer, neuroblastoma) o Substrates ▪ Substrates include numerous anticancer drugs (e.g., vincristine, etoposide, anthracyclines, methotrexate), opiates, antidepressants, statins, antibiotics ▪ Natural substrates include pro-inflammatory molecules (e.g., leukotriene, C4), hormones, antioxidants (including glutathione) BIOL2174: Cell Physiology – Lecture Notes o Humans have 13 ABCCs, at least 9 of which have bee found to efflux drugs from cells ABCC1 – structure ABCC1 bound to LTC4 LTC4 is produced by immune cells as part of the inflammatory response LTC4 consists of an arachidonic acid derivative (black) conjugated to the tripeptide glutathione (red) The binding site has a positively charged region that coordinates the glutathione moiety (P pocket) and a largely hydrophobic area to accommodate the lipid tail (H pocket) BIOL2174: Cell Physiology – Lecture Notes ABCC1 – can the substrate binding site explain substrate diversity The H pocket is larger than the lipid tail of LTC4 – could accommodate other hydrophobic moieties o Polar side chains in the H pocket could interact w/ polar functional groups found on otherwise hydrophobic substrates Many residues in binding site alter side-chain positions upon binding to LTC4 – suggests plasticity in the binding site that allows for adaptability to varying substrates Glutathione is a known substrate despite only filling P-pocket o They fact that the P-pocket comprises both TM bundles means that just filling the P- pocket is enough to bring the 2 halves of the transporters together ABCC1 mechanism Local and global conformational changes are induced by substrate binding The NBDs are closer together when substrate is bound The substrate serves as a connecting bridge to bring the 2 halves of the transporter together ATP hydrolysis occurs when the 2 NBDs dimerise to form a complete catalytic site – this is more likely to occur when substrate is bound ABC transporters involved in resistance to anticancer drugs P-glycoprotein (aka ABCB1) Multiple isoforms of Multidrug Resistance-associated Proteins (MRPs; aka ABCCs) Breast Cancer Resistance Protein (BCRP; aka ABCG2) BIOL2174: Cell Physiology – Lecture Notes ABCG2 in resistant cancers ABCG2 – originally identified in a multidrug-resistant breast cancer line that did not overexpress ABCB1 or ABCC1 o IT has been found and its increased expression correlated to multidrug resistance phenotypes, in numerous types of cancer cells o Substrates include a wide variety of anticancer drugs, sulfate, glucuronide conjugates of sterols and xenobiotics, and various natural compounds, toxins, fluorescent dyes and antibiotics ABCG2 – structure ABCG2 – structure and mechanism ATP binding induces conformational changes that collapse the substrate binding cavity; substrate is squeezed out Findings suggest ATP binding is sufficient of substrate extrusion; ATP hydrolysis might be required to reset the transporter to an inward facing conformation BIOL2174: Cell Physiology – Lecture Notes Comparing ABCB1, ABCC1 & ABCG2 ABC transporters can confer multidrug resistance in microbes too Multidrug efflux systems don’t have to be ABC transporters Multidrug efflux system = any protein/protein complex that expels more than one drugs from its site of action Often, they are on the cell plasma membrane and transport the drug out of the cell, but there are exceptions o E.g., efflux of drug from an organelle in which the drug acts o E.g., pumping drugs that act in the cytosol into an organelle (e.g., P, falciparum Multidrug Resistance 1) BIOL2174: Cell Physiology – Lecture Notes Multidrug efflux systems in bacteria Sometimes the main mechanism of resistance; sometimes an initial mechanism that buys the bacteria time to come up w/ their main mechanism 6 transporter families have members involved in multidrug efflux in bacteria 1. Major Facilitator Superfamily (MFS) 2. Small Multidrug Resistance (SMR) family 3. Multidrug and Toxic compound Extrusion (MATE) family 4. Resistance-Nodulation-cell Division (RND) superfamily 5. Proteobacterial Antimicrobial Compound Efflux (PACE) family 6. ATP-Binding Cassette (ABC) transporter superfamily Some have roles other than drug efflux, e.g., in: o Virulence (e.g., by secreting antimicrobial peptides) o Cell-to-cell communication (e.g., by exporting signally molecules involved in quorum sensing) o Formation of biofilms o Lipid transport o Transport of noxious compounds encountered in natural environment (e.g., bile salts) Antibiotic resistance can arise from an increase in intrinsic efflux activity – through e.g., overexpression, asymmetric accumulation during division, or mutation of genes encoding the transporters Gram-negative bacteria have a cell envelope comprising 2 membrane – tripartite efflux systems assemble to span the envelope Structural data exist for members of each bacterial multidrug efflux family except for the PACE family BIOL2174: Cell Physiology – Lecture Notes Drug resistance The problem Resistant bacteria o To assess bacterial susceptibility to different antibiotics (which diffuse out of the disks) – there is a zone of inhibition (an absence of bacterial growth) around effective antibiotics Resistant parasites o E.g., Plasmodium, giardia, trichomonas o Emerging resistance to antimalarial artemisinin-based combination therapies Resistant fungi o E.g., Candida, Aspergillus o A growing threat o Multidrug-resistant Candida infections are particularly problematic Resistant cancer cells o A major reason behind chemotherapy failure Why so much resistance Rapidly proliferating cells have a lot of opportunity for genetic change If a genetic change confers a survival advantage in the presence of the drug, it will be selected for Drug discovery Drug target must be essential, inhibitable, & accessible Selectivity What sets cancer apart? o Rapid proliferation phenotype o Different expression pattern for kinases Most anticancer drugs have a ‘narrow therapeutic window’ (i.e., the dose required for clinical benefit is close to a dose that will cause too much toxicity) – this means that even a low level of resistance is problematic The mechanism – how antimicrobial drugs work BIOL2174: Cell Physiology – Lecture Notes Mechanisms of drug resistance Key drug away from its target o Reduce entry of drug into cell/compartment containing target o Efflux drug from cell/compartment containing target o Sequester drug in a compartment not containing target o Alter cell physiology to reduce drug accumulation in cell/compartment containing target Change the drug o Decreased activation of drug o Inactivate drug ▪ Degrade ▪ Modify Change the drug’s target o Mutate the target o Overexpress the target Repair the drug-induced damage Stall until the drug is gone BIOL2174: Cell Physiology – Lecture Notes Lecture 23 – Drug resistance Drug resistance cont. Decreased activation of drug Many anticancer drugs must be activated in order to become efficacious Drug resistance can result from decreased drug activation o E.g., resistance of human leukemic cells to cytarabine (cytosine arabinoside; AraC) ▪ Cytarabine is on the WHO’s list of essential medicines. It is used in the treatment of acute myeloid leukemia, acute lymphocyte leukemia, chronic myelogenous leukemia, and non-Hodgkins lymphoma AraC (a nucleoside analogue) is activate by conversion to AraC-triphosphate by sequential phosphorylation events carried out by deoxycytidine kinase, dCMP kinase, and nucleoside diphosphate kinase o AraC-triphosphate is incorporated into DNA and RNA, resulting in cytotoxicity o Rapidly dividing cells (undergoing DNA replication) are most affected o Resistant cells have mutated or downregulated deoxycytidine kinase have been reported o Other mechanisms of AraC resistance not related to decreased activation also exist Mutate the target Examples are easy to find – a common mechanism if there is a single protein target E.g., topoisomerase II targeting anticancer drugs o Multiple clinically used anticancer drugs target the enzyme topoisomerase II (e.g., doxorubicin, daunorubicin, etoposide, amsacrine) o Topoisomerase II inhibitors are used to treat a wide variety of cancers, including solid tumours and blood cancers o Topoisomerase II complexes w/ DNA (transiently) and detangles it, allowing DNA replication to take place o E.g., resistance of human leukemia cells to amsacrine can be mediated by a mutation in topoisomerase II E.g., HER2 targeting anticancer drugs o ~25% of breast cancers overexpress the tyrosine kinase “human epidermal growth factor receptor 2” (HER2) o HER2 initiates several signalling pathways that promote proliferation and oppose apoptosis o HER2 overexpression is associated w/ aggressive disease and poor prognosis o There are specific therapies used clinically to target HER2 – trastuzumab (Herceptin) and lapatinib o These therapies have clear clinical benefits, but many patients relapse o Some causes of resistances to HER2-targeted therapies result form mutations in HER2 that reduce drug binding Repair the drug-induced damage Several anticancer drugs work by directly or indirectly damaging DNA DNA damage response mechanisms can reverse the drug-induced damage Cisplatin-containing combination chemotherapy is used in the treatment of many cancers BIOL2174: Cell Physiology – Lecture Notes o The responsiveness is initially high but many cancer patients eventually relapse w/ cisplatin-resistant disease o Cisplatin binds to DNA and causes damage (e.g., crosslinking) and mutations – in sensitive cells this prevents DNA replication Cells have mechanisms to recognise and repair DNA damage – if the damage is too extensive apoptosis is normally induced 2 major repair pathways are mismatch repair and nucleotide excision repair There are examples of cisplatin-resistant cancer cells that show increased expression of genes associated w/ both the nucleotides excision repair and mismatch repair pathways Other cisplatin resistance mechanisms, including reduced accumulation of drug, have also been reported In-vitro evolution Developing assays to study protein targets Depends on what sort of protein o E.g., add the substrate and detect the product o E.g., measure ATP hydrolysis (using a colorimetric reaction to detect phosphate) o E.g., measure the conversion of NAD+ to NADH (colorimetric assay) What if you have no good clues about what the protein might do? o Untargeted metabolomics ▪ Are the levels of any metabolites different? (+drug compared – drug) Combating resistance Reversing resistance BIOL2174: Cell Physiology – Lecture Notes Designing drugs w/ resistance in mind Exploiting collateral hypersensitivity Combing drugs Reversing resistance Reversing ABCB1-mediated resistance → why is reversing multidrug resistance via ABCB1 inhibition so challenging? o It is difficult to inhibit ABCB1 fully while avoiding toxicity o In many cancers other multidrug efflux systems or other resistance mechanisms play important roles Designing drugs w/ resistance in mind What features can make drugs less resistance prone? o Drugs that evade multidrug efflux systems o Drugs w/ more than one target (polypharmacology) o Drugs that kill cells rapidly o Drugs that target a non-protein-mediated process (e.g., chloroquine) Exploiting collateral hypersensitivity In order to become resistant Drug X, a cell had to change in some way That change resulted in the cell becoming more sensitive to Drug Y) BIOL2174: Cell Physiology – Lecture Notes Many different mechanisms o E.g., for 2 drugs that bind to the same protein target, a mutation might change the structure of the protein so that Drug X binds worse (giving rise to Drug X resistance) but Drug Y binds beter (yielding hypersensitivity to Drug Y) Expressing ABCB1 makes cancer cells more sensitive to a range of drugs Researchers are pursuing compounds that eliminate ABCB1-expressing cells, to be used in conjunction w/ traditional chemotherapy A variety of mechanisms have been proposed for different compounds, e.g., Combining drugs Combination therapies (i.e., treatments combining 2 or more therapeutic agents) are now commonly used to treat diverse diseases The use of combinations therapies reduces the probability of resistance development o 1 in 109 cells acquires a mutation that renders it resistant to Drug X o 1 in 109 cells acquires a mutation that renders it resistant to Drug Y o What is the probability of a cell simultaneously acquiring resistance to Drug X and Drug Y → 1 in 1018 What factors should be considered when deciding which drugs to combine o Pharmacokinetics – don’t want the drugs to spend time in the body an monotherapies o Drug interactions – don’t want to combine drugs that will antagonise each other o Off-target effects of drug combination – need to avoid toxicity o Different resistance mechanisms – don’t want the drugs to succumb to the same resistance mechanism o Exploit collateral hypersensitivity if possible to further reduce the probability of resistance emergence o Onset of action – fast for at least one drug – important for infectious diseases where rapid reduction of microbe burden is required o Formulation – combine in same preparation so one drug is not taken alone, with the other held onto for later BIOL2174: Cell Physiology – Lecture Notes Lecture 24 – pH regulation 1 pH in cell cytosol pH inside cells If H+ ions were at electrochemical equilibrium across the cells plasma membrane then their distribution would be in accordance with the Nernst equation o Mammalian system – normal extracellular pH = 7.4; membrane potential (Em) = - 60mV o Putting these values into the Nernst equation predicts an intracellular pH of 6.4 BIOL2174: Cell Physiology – Lecture Notes For most cells the pH in the cytosol (pHi) is usually about 7.2 o The pH in the cytosol of most cells is higher than that predicted if H+ ions were at equilibrium across the cell plasma membrane o Conclusion – the cell has mechanisms for actively regulating its cytosolic pH Measuring cytosolic pH Load cells w/ a pH-sensitive fluorescent dye Transfect cells w/ a pH-sensitive protein Measuring cytosolic pH – fluorescent dye Starts w/ acetoxymethyl (AM) ester form – membrane permeant, will enter cells Inside cells – ester groups cleaved off by esterases Yields fluorescent molecule that is charged, much less membrane permeant Wash cells to remove extracellular dye Radiometric measurements o In the case of BCECF, obtain the ratio of the fluorescence emanating from excitation at ~500 nm (pH-sensitive) to that measured on excitation at 440 nm (pH-insensitive) o To calibrate – suspend cells in calibration salines of known pH, add on ionophore that will force the intracellular pH to become the same as the extracellular pH BIOL2174: Cell Physiology – Lecture Notes Techniques used to study intracellular pH Perturbing pH using NH4Cl o Cells are exposed to an external solution containing NH4Cl o The NH4+ ions is in equilibrium w/ low levels of NH3 in the solution o The NH3 being uncharged, enters the cell far more rapidly than the NH4+ (Step 1 below) o Once inside, the NH3 combines w/ a H+ ion from within the cell to re-establish the NH4+/NH3 equilibrium o This has the effect of alkalinising the cell (step 2) o NH4+ permeates the cell membrane, typically via K+ channels, and enters the cell, driven by the (inside negative) membrane potential (step 3) o This has the effect of bringing H+ ions into the cell resulting in a decline in the pHi from its new alkaline value o Under these conditions, upon removal of external NH4CL then NH3 will come rushing out of the cell o Within a very short time the NH4+ will dissociate (Step 2 in reverse) and the resultant NH4 will leave the cell, leaving the H+ ion behind (Step 1 in reverse) o This results in an immediate acidification of the cell cytosol, with pHi ‘undershooting’ its original value o The cell responds to the imposed acidification by activating ‘acid extruding’ pH regulatory mechanisms (step 5) BIOL2174: Cell Physiology – Lecture Notes Importance of pH regulation Why is pH regulation important pH affects: o the charge on ionisable groups on proteins and thereby protein function o Metabolism o The state of polymerisation of the cytoskeleton o The activity of transport proteins pH regulation is an essential process H+ coupled transporters Most of the secondary transporters in humans and higher eukaryotes use the Na+ electrochemical gradient to drive transport However, there are examples of human secondary active transporters that use the H+ electrochemical gradient to energise transport The uptake of small (di-, tri- and tetra-) peptides (originating from dietary protein) into intestinal and kidney epithelial cells is mediated by Na+-independent, H+-dependent transporters hPepT1 and hPepT2 are human H+:peptide transporters A no. of drugs, including some antibiotics, antivirals and Anticancer drugs, are peptide-like, and use these transporters to be taken up into the body Uptake of peptides in the small intestine Xenopus oocytes Commonly used heterologous expression system for the study of transport proteins Large cell into which you can inject RNA The oocyte will translate the injected RNA into protein Low endogenous transport activity (therefore, low background) BIOL2174: Cell Physiology – Lecture Notes Transporters involved in regulating cytosolic pH Transporters involved in pH regulation Transporters involved in pH regulation usually transport either H+, OH- or HCO3- ions Transporting HCO3- ions is equivalent to transporting OH- ions as the following reaction takes place A HCO3- ion, like an OH- ion, has the ability to soak up or neutralise a H+ ion Transporting H+ ions out of a cell, or OH- of HCO3- ions into a cell makes the cell more alkaline (i.e., increases the pH) o Transporters that do this are called acid extruders Transporting H+ ions into a cell, or OH- or HCO3- ions out of a cell makes the cell more acidic (i.e., lowers the pH) o Transporters that do this are called acid loaders BIOL2174: Cell Physiology – Lecture Notes Lecture 25 – pH regulation 2 Transporters involved in regulating cytosolic pH Transporters involved in regulation of intracellular pH A transporter which extrudes alkali from the cytosol – i.e., mediates recovery from an intracellular alkalinisation o Cl-/HCO3- exchanger ▪ Mediates 1:1 exchange of Cl- from HCO3- (typically HCO3- moving out of the cell in exchange for Cl- moving into the cell) ▪ Several isoforms – AE1-AE3 (AE stands for Anion Exchanger) AE1 is the so-called band 3 anion transporter of red blood cells AE2 is the housekeeping anion exchanger and is widely distributed Distribution of AE3 is more restricted ▪AE2 is activated by cellular alkalinisation and returns pHi to normal by extruding base (HCO3-) and thereby reacidifying the cell AE2 may possess an internal allosteric regulatory site that activates the exchanger at alkaline values of pHi o Na+/H+ exchangers (NHEs) ▪ Responds to cellular acidification by extruding 1 H+ ion in exchange for the influx of 1 Na+ ion ▪ Inhibited by amiloride ▪ There is currently known to be at least 9 isoforms of the Na+/H+ exchanger that catalyse electroneutral transport – NHE1-NHE9 NHE1 is the housekeeping version and is present in almost all cell- types BIOL2174: Cell Physiology – Lecture Notes The others are less widely distributed ▪ There is also a separate clade of Na+/H+ antiporters containing members believed to catalyse electrogenic transport (i.e., not a 1:1 exchange of Na+ and H+) – NHA1-2 ▪ Crystal structure solved for a Na+/K+ exchanger from Thermus thermophilus (21% sequence identity to human NHA2) ▪ CHP1 important for: Biosynthetic maturation Localisation to cell surface pH sensitivity ▪ Na+/H+ exchange is activated by a variety of substances including hormones, NTs, growth factors ▪ Na+/H+ exchange is also activated by cell shrinkage as part of the cell’s volume-regulatory response ▪ Hyperactivation of Na+/H+ exchangers during ischemia-reperfusion episode (e.g., heart attack or stroke, followed by recovery of normal blood flow) disrupts the intracellular ion balance and leads to cardiac or neuronal dysfunction and damage The role of the Na+/H+ exchanger in ischemia-reperfusion injury NHE – role in ischemia-reperfusion injury If the blood flow to the heart or brain is blocked the cells are starved of oxygen and mitochondrial ATP production (which is reliant on oxygen) ceases Glycolysis is increased to try to produced sufficient ATP through this pathway The increase in glycolysis generates large quantities of lactic acid in the cytosol but is unable to keep the cell adequately supplied with ATP, so there is a progressive decrease in the ATP concentration The combined effect of the production of excess lactic acid and the net hydrolysis of ATP is to generate a large intracellular acid load (i.e., excess H+ ions in the cytoplasm) This leads to activation of the Na+/H+ exchanger and there is a net efflux of H+ ions coupled to a net influx of Na+ ions The Na+ ions entering the cell via the Na+/H+ exchanger would normally be pumped out by the Na+/K+ pump, but w/ ATP levels so low the pump does not work properly and there is a build-up of Na+ in the cytosol Cardiac cells and neurons both have in their plasm membrane a Na+/Ca2+ exchanger which usually works to extrude Ca2+ form the cytoplasm, but with the increasing cytoplasmic Na+ concentration this exchanger begins to catalyse the net influx of Ca2+ In heart cells the accumulation of cytosolic Ca2+ leads to arrhythmias and myocardial stunning In neurons a sustained increase in cytosolic Ca2+ also causes serious cell damage BIOL2174: Cell Physiology – Lecture Notes Nor does it get better once normal blood flow is restored After a period of ischemia the cytosolic pH is substantially lower than it should be Once blood flow is restored and all the H+ ions, lactic acid etc., are washed away from the fluid surrounding the cells, the intracellular pH is much lower than the extracellular pH There is therefore, a net outward H+ gradient and this causes the Na+/H+ exchanger to