LEC2 - Electrical Properties, Signalling - Fundamentals of Human Physiology PDF

Fundamentals of Human Physiology Spring 2019 Session 2.1 – Cell Physiology Electrical Properties of the Cell and the Action Potential Patricio E. Mujica, Ph.D. Department of Pharmacology, Physiology and Neuroscience [email protected] 1 Objectives By the end of this lecture you should be able...

Fundamentals of Human Physiology Spring 2019 Session 2.1 – Cell Physiology Electrical Properties of the Cell and the Action Potential Patricio E. Mujica, Ph.D. Department of Pharmacology, Physiology and Neuroscience [email protected] 1 Objectives By the end of this lecture you should be able to: Define the term membrane potential List the intracellular and extracellular concentrations of Na+, Ca2+, K+ and Cl– ions Understand and describe the role of concentration gradients and permeability in the development of the resting membrane potential (especially Na+ and K+) Understand the concept of equilibrium potential for a given ion, and use the Nernst equation to calculate it. Identify the Goldman-Hodgkin-Katz equation and be able to use it in the calculation of the resting membrane potential Define the term electrochemical gradient and be able to identify the forces involved in ion movement (chemical vs. electrical) Understand and define the following terms: depolarization, repolarization, hyperpolarization, inward current, outward current and refractory period Define and describe the events that occur in the neuronal action potential Identify the role of the refractory period and its phases on the neuronal action potential Describe how myelination increases the conduction velocity of axons The cell membrane imposes a barrier to the diffusion of polar and charged molecules Constituents of the cell membrane according to the fluid mosaic model: Phospholipids Proteins (integral, peripheral) Glycoproteins Glycolipids Cholesterol Phospholipids are amphipathic molecules X+ Intracellular and extracellular concentrations (mM) of physiologically relevant ions Ion Outside Y– Outside Inside Cations Na+ 145 15 ~9.7 K+ 5 150 30 Ca2+ 1 0.0001 10,000 Anions extracellular intracellular Inside Cl– 108 10 10.8 HCO3– 24 8 3 OA– 0 155 1/∞ Transport processes create a measurable separation of charges at the cell membrane that translates into a membrane potential Active transport processes utilize ATP to pump ions against their concentration gradient. This movement leads to the separation of electrical charges at the cell membrane. Cations are pumped out of the cell and create a net positive charge on the extracellular side of the membrane, while the intracellular side becomes negatively charged. This movement of ions cannot be reversed by simple diffusion Ion movement establishes the ion concentration differences seen before. Concentration differences (in/out) stabilize at steady-state No net movement of ions Influx = Efflux Result of active transport + leak Steady state ion concentrations create an electrical potential difference This is the cell’s membrane potential Can be measured with electrodes The cell’s membrane potential can be measured using electrodes Membrane potential (Vm): the voltage difference that exists between the inside and outside of the cell Vm = Vo – Vi Vm: membrane potential difference Vo: extracellular voltage, measured with reference electrode Vi: intracellular voltage, measured with intracellular electrode 5 Ions move across the cell membrane according to their electrochemical gradient, creating ionic currents Two forces determine the likelihood and the direction of ion movement across the cell membrane: The ion’s concentration gradient. The ion’s electrical gradient, or electrical potential, between the inside and the outside of the cell. For all practical purposes, the ions relevant for our understanding of the cell’s electrical properties and the action potential are K+, Na+, Ca2+ and Cl– extracellular Na+ K+ Ca2+ Cl– +++++++++++++++++ – – – – – – – – – – – – – – intracellular Na+ K+ Ca2+ Cl– Ions cannot simply diffuse across the membrane: they need pathways provided by ion channels Boron & Boulpaep, Medical Physiology (2nd ed, 2009) Ions move across the cell membrane according to their electrochemical gradient, creating ionic currents The net movement of an ion across the membrane generates an ionic current The flux of an ion X+ (the amount of ion that moves across the membrane per unit time) depends on three factors: The concentration gradient (EX) The membrane potential (Vm) The ion membrane conductance (gX) The net flux of an ion through the membrane can be understood as an electrical current (Ix), which can be determined by Ohm’s law: IX = gX (Vm – EX) In this equation, (Vm – EX) is the driving force for the movement of ion X+ gX is the membrane conductance, specific for X+ Boron & Boulpaep, Medical Physiology (2nd ed, 2009) Ions move across the cell membrane according to their electrochemical gradient, creating ionic currents From IX = gX (Vm – EX) where (Vm – EX) = driving force for the movement of ion X+ gX = membrane conductance, specific for X+ and related to the presence of permeation pathways (ion channels) on the plasma membrane An inward current results when there is an influx of positive charges (i.e. Na+, Ca2+) or an efflux of negative charges An outward current results when there is an efflux of positive charges (i.e. K+) or an influx of negative charges Na+ K+ Ca2+ Cl– gNa gK gCa gCl Na+ K+ Ca2+ Cl– extracellular intracellular What if the net current for an ion is zero? IX = 0 means either gX = 0 or (Vm – EX) = 0 In general, gX is a nonzero value, but if (Vm – EX) = 0 then Vm = EX The equilibrium potential for ion X (EX) is the membrane potential Vm at which the net movement of ion X is zero Also known as reversal potential Zero net movement of an ion means that the ionic current IX is zero, and ion X is said to be at equilibrium Ionic currents are driven by a quantifiable electrochemical potential: the Nernst equation We can understand electrochemical potentials as the combination of driving forces (chemical potential energy, electrical potential energy) that determine the movement of an ion across a membrane The total driving force (potential energy) ∆ X for the movement of an ion X can be expressed as: ∆ X= [ ] + [ ] X R = ideal gas constant T = temperature zX = charge of ion X F = Faraday’s constant where Since equilibrium is a thermodynamic state at which potential energy is zero, then ∆ [ ] 0 = [ ] + X = 0 and therefore: X Solving for Vm, we can determine the membrane potential value at which ion X is at thermodynamic equilibrium: =− [ ] [ ] X = X or this expression is the Nernst equation 𝑡 𝑢 𝑡 𝑢 𝑜 𝑡 𝑋 𝑢 𝑋 𝐹 𝑋 𝑧 𝑜 𝑜 Transforming ln to log10 and assuming standard values for R, F and T = 37 ºC: 𝑚 𝑉 𝐹 𝑉 𝑧 𝐹 𝑛 𝑛 𝑖 𝑛 𝑇 𝝁 𝝁 𝑋 𝑋 𝑋 𝐸 𝑅 𝑖 𝑖 𝑧 𝑛 𝑙 𝑛 𝑙 𝑇 𝑛 𝑙 𝑅 𝑇 𝑅 𝜇 𝑉 𝑚 𝑚 (Note that the negative sign in the equation above can be eliminated by applying logarithm properties) Ionic currents are driven by a quantifiable electrochemical potential: the Nernst equation We can use the Nernst equation to determine the membrane potential at which individual ions achieve equilibrium  their equilibrium potential Considering the values for Eion on the table: What is the membrane potential at which sodium ions will stop moving across the membrane? How about potassium? Calculate the reversal potential for calcium (zCa = 2) at 37 ºC. A cell’s resting membrane potential is determined by the tendency of each ion to achieve equilibrium The resting membrane potential (RMP) is the membrane potential difference for a cell at rest This concept is especially important for excitable cells such as neurons, skeletal muscle cells and cardiomyocytes The RMP is established by the charge and concentration of all ions that can move across the membrane Remember: IX = gX (Vm – EX)  electrochemical gradients and membrane conductances determine ionic currents Each ion attempts to drive the membrane potential toward its own equilibrium potential Different cell types have different membrane potentials: Skeletal and cardiac muscle = –85 mV Sensory/motor neurons = –70 mV Smooth muscle = –60 mV Red blood cells = –10 mV When the cell is at rest, RMP is closest to the equilibrium potential for K+ (EK = –90 mV) RMP is far from equilibrium potentials for Na+ (ENa = +60 mV) and Ca2+ (ECa = find out!) because the permeability of these ions is low at rest Potassium ions are the biggest contributors to the resting membrane potential Potassium leak channels allow the efflux of potassium at rest and potassium ions will continue to flow until the cell reaches the potassium equilibrium potential (-90mV) Cells possess sodium leak channels that allow a small influx of sodium ions, the cell never reaches the potassium equilibrium potential Determination of the resting membrane potential: the Goldman-Hodgkin-Katz (GHK) equation While potassium ions are the biggest contributors to the resting membrane potential, other ions contribute as well At rest, the cell membrane is also permeable to sodium and chloride Ca2+ Cl– Na+ K+ gNa gK gCa gCl Na+ K+ Ca2+ Cl– extracellular intracellular Calcium plays a negligible role in the resting membrane potential of excitable cells, but it is crucial when excitation takes place on several cell types. The Goldman-Hodgkin-Katz equation (a.k.a. constant field equation) considers the contribution of each permeant ion (i.e. ion that can cross the membrane) in terms of its membrane permeability (Pion). Action potentials occur in excitable cells Action potentials are the basic mechanism for transmission of information in excitable cells (nerve and muscle cells) Electrical activation of a cell that involves brief changes in the membrane potential: depolarization and repolarization Result of the changes in membrane permeability of specific ions that move into or out of the cell via their respective ion channels Most action potentials (in nerve and skeletal muscle) are due to Na+ and K+ movement Cardiac cell action potentials also involve the influx of Ca2+ APs in neurons and skeletal muscle cells are very fast ~1-2 millisecond (ms) Cardiac cells have APs that last for 250-400 ms Action potential terminology Depolarization: making the membrane potential less negative Repolarization: making the membrane potential more negative (brings the membrane potential back to the resting value) Hyperpolarization: making the membrane potential more negative (usually more negative than the resting membrane potential) Threshold potential: the membrane potential at which the action potential will fire An inward current is necessary to bring the cell to threshold Overshoot: the portion of the AP where the Vm >0 Hyperpolarizing afterpotential (undershoot): the portion of the AP where Vm is more negative than RMP Refractory period: a period during which another normal action potential cannot be elicited Neurons respond to stimulation with graded potentials, which may become action potentials The neuronal action potential Changes in Na+ conductance during the neuronal action potential depend on the gating mechanism of voltage-gated sodium channels The refractory period Defined as the time required for Na+ channels to recover from inactivation They must become available to be opened again Activation and inactivation gates must reset back to their resting positions Requires the repolarization of the cell membrane During this recovery period, the neuronal membrane is resistant to stimulation No normal action potential can be generated The refractory period can be divided into two segments Absolute refractory period: no stimulus, regardless of its magnitude, can stimulate an action potential Relative refractory period: when a larger than normal stimulus can develop an action potential (usually abnormal in shape) Action potential propagation and conduction velocity: the role of axon diameter Two factors affect the conduction of an action potential of a neuron Axon diameter Axon membrane resistance (to ion leakage) Large diameter axons (i.e. giant squid axons ~1mm) have a low resistance to ion flow Ion channels have to remain open constantly for action potentials to be conducted down the entire axon Small neurons have a higher resistance to ion flow They also show greater ion leak out of the axon, leading to poor AP conduction In the mammalian nervous system the small axons are insulated by a myelin sheath to minimize ion leak out of the axon Action potential propagation and conduction velocity: the role of axon myelination Myelination is the lipid insulation of axons by the membranes of accessory cells Oligodendrocytes in CNS Schwann cells in the PNS Axon myelination the increases membrane resistance to ion leakage Current must flow through nonmyelinated, low-resistance sections: nodes of Ranvier Na+ and K+ channels are clustered at the nodes of Ranvier Membrane resistance is low and action potentials can occur APs “jump” from node to node (saltatory conduction), which increases the conduction velocity (as compared to a non-myelinated axon) Presence or absence of myelin gives rise to saltatory vs. continuous conduction of action potentials 4 6 5 Express Local t 1 t S 25 11 t 6S S 6 8 11 t S 0 1 S 03 t 9 t S 6 5 t S 7 7 6 t S 8 t S 9 Classification of mammalian axons by conduction speed Parameter Type A Type B Type C Speed of conduction Fastest (250 mph) Slow (32 mph) Slowest (1–5 mph) Myelinated Yes Mostly No Largest Intermediate Smallest (5–20 micrometers) (2–3 micrometers) (0.5–1.5 micrometers) Sensory fibers; motor neurons Efferent autonomic fibers; some sensory Visceral sensory; some efferent autonomic fibers; specific somatosensory pathways (pain, temperature, pressure) Skeletal muscle, joints Internal organs Internal organs, skin Diameter Function Innervation Fundamentals of Human Physiology Spring 2019 Session 2.2 – Cell Physiology Principles of Cell Signaling Patricio E. Mujica, Ph.D. Department of Pharmacology, Physiology and Neuroscience [email protected] Objectives By the end of this lecture you should be able to: Define the four methods of local communication between cells Gap Junctions Contact dependent signals Autocrine signaling Paracrine Signaling Compare and contrast the long-distance modes of communication of the body Endocrine Signaling Nervous System Signaling Understand the concepts of ligand and receptor in the context of cell-cell communication Describe the general signaling mechanisms for the different classes of receptors Intracellular receptors (cytosolic and nuclear) Membrane receptors (ligand-gated ion channel, receptor-enzyme, G protein-coupled receptors, and integrins) Understand and illustrate the specific signaling mechanisms for the GPCR-adenylyl cyclase-cAMP system and the GPCR-phospholipase C - IP3/DAG - Ca2+/PKC system Define the concepts of specificity, saturation, and competition to the understanding of cell signaling processes. Local communication coordinates cellular activity over short distances Gap Junctions: several connexin proteins assemble in connexons which directly bridge the cytoplasm of adjacent cells Contact-dependent signals: occur by the direct interaction between two cells – allow the transfer of small molecules (amino acids, ATP etc.) and electrical signals (ions) from cell to cell – A surface molecule on one cell binds to the receptor on another cell – Important role in the conduction of action potentials of cardiac muscle – Important in the immune system and play a role in the control of cell cycle and cell-adhesion processes Autocrine signaling occurs when a chemical signal acts on the same cell that released it. Paracrine signaling occurs when a chemical signal is released and it acts on neighboring cells (near the site of release) Many paracrine signaling mediators (histamine, cytokines, etc.) Long distance modes of cell communication The endocrine system uses hormones (chemical signals) that are released into the blood to act on specific receptors located on cells in many places in the body. The nervous system uses both chemical signals (neurotransmitters, neurohormones) and electrical signals (action potentials) to communicate with cells and specific cell receptors in the body. Ligands and receptors Ligand Ligand cannot bind receptor B No signaling process activated Cell membrane Receptor A Receptor B Ligand-receptor complex formed Activation of intracellular signaling process The chemical nature of a ligand determines the receptor it binds to Intracellular receptors are ligand-sensitive transcription factors and modify cell behavior through changes in gene expression Ligands: Steroid hormones (estrogen, testosterone, progesterone) Vitamin D Retinoic acid and derivatives Receptor held in inactive form by chaperone proteins Ligand binding decreases receptor affinity for inhibitory proteins and induces conformational changes: Binding of coactivators Nuclear translocation Unmasking of DNA-binding domains Membrane-bound receptors Signal transduction pathways of membrane-bound receptors and the role of signal amplification Receptors Tyrosine Kinase (RTKs) are ubiquitous members of the enzyme-linked receptor family Sequence of RTK activation (1) (4) Tyrosine kinase signaling is an important component of insulin, growth hormone and insulin-like growth factor-1 actions on the body (2) (3) (5) G protein-coupled Receptors (GPCRs) comprise a large superfamily of seven-transmembrane receptors https://katritch.usc.edu/research.html Gacasan et al, 2017 AIMS Biophysics https://proteopedia.org/wiki/index.php/G_protein-coupled_receptor Nearly 800 genes in the human genome Receptors are coupled to intracellular heterotrimeric GTP-binding (G) proteins Alpha subunit determines receptor coupling to intracellular signaling modules Four general types based on alpha subunit: Gq, Gs, Gi/o, G12/13 Beta-gamma complex can also signal independently of alpha subunit Gq-coupled signal transduction is mediated by phospholipase C (PLC) and leads to Ca2+ release from intracellular stores Gq signaling pathway The Gq signaling pathway is activated in a variety of physiological processes: activation of the sympathetic nervous system via α-adrenergic receptors Facultative water reabsorption in the kidney by vasopressin (antidiuretic hormone) Regulation of blood pressure and sodium homeostasis by angiotensin Gs-coupled signal transduction involves activation of membrane-bound adenylyl cyclases and synthesis of intracellular cAMP The Gs signaling pathway is activated in a variety of physiological processes: activation of the sympathetic nervous system via -adrenergic receptors 𝝱 Transduction of olfactory stimuli in the olfactory mucosa Signaling pathways can be modulated in several ways: ligand binding, selectivity, specificity Some ligands have many receptors One ligand can bind to many different receptor types The type of response is specific to the receptor that it binds to (i.e. epinephrine binds to two different sympathetic adrenergic receptors) Protein binding characteristics can affect how the ligand binds to its receptor: Specificity: a specific ligand binds to a specific receptor Competition: multiple ligands can bind to the same receptor, but some can bind at a higher level (affinity) than others Saturation: all of the of binding sites for a specific ligand have been occupied and no more of the ligand can bind (protein activity reaches a maximum rate) Agonists: ligands that bind to and activate the receptor and cause a response Response may be maximal (full agonist), partial (partial agonist) or in the “opposite direction” (inverse agonist) Antagonists: ligands that bind to the receptor and no response occurs Prevent an agonist (of any kind) from triggering a response 36 Signaling pathways can be modulated in several ways: receptor up/down-regulation, receptor desensitization High concentrations of a signaling molecule can down-regulate a receptor Down-regulation is a decrease in the number of receptors in the membrane for a signaling molecule It occurs to prevent an enhanced response and maintain physiological conditions Receptor desensitization can also occur with elevated levels of signaling molecules It suppresses the activity of the ligand by a chemical modulator binding to the receptor which blocks the activation of the receptor Clinically, down-regulation and desensitization can cause a lower or less effective response from the cell signaling May occur when patients have taken a drug for an extended and continuous period Partially explains the development of addictive behaviors due to changes in brain receptor biochemistry Up-regulation of the receptor occurs when ligand levels have decreased This is an attempt to enhance the response when the levels are low to maintain the normal physiological responses in the body