Transmembrane Transport of Ions and Small Molecules - PDF

1 TRANSMEMBRANE TRANSPORT OF IONS AND SMALL MOLECULES 10/6/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student Learning Outcomes ▪ Distinguish molecules that can diffuse thro...

1 TRANSMEMBRANE TRANSPORT OF IONS AND SMALL MOLECULES 10/6/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student Learning Outcomes ▪ Distinguish molecules that can diffuse through a lipid bilayer from those that require transporters to cross a membrane. ▪ Explain the mechanism by which cells maintain the concentration differences of small molecules across biological membranes. ▪ Compare and contrast passive vs active transport (and between primary and secondary active transport). ▪ Compare and contrast different kinds of channels (including gated channels), transporters/carriers, and pumps. ▪ Explain the role of membrane potential and ion channels in action potentials.  3 Lecture Structure 1. Selective permeable membrane 1.A. Concentration and electrochemical gradients 1.B. Transport Mechanisms 2. Passive transport 2.1. (Diffusion) 2.2. Facilitated a) Transporters/carriers b) Channels & 3. Active transport 3.1. Primary Active Transport 3.2. Secondary Active Transport 4. Action Potential and synapse  4 1. Selective permeable membrane ▪ Impermeable to hydrophilic molecules ▪ Large, polar molecules ▪ Ions ▪ Permeable (simple diffusion) – small, non-polar molecules (gases) ▪ Slightly permeable – water Selective transport: ▪ Determines cytoplasm composition ▪ Transport rate: ▪ Differences in charge ▪ Differences in concentration  5 1.A. Concentration gradients vs electrochemical gradient Gradients = potential energy (stored energy) ▪ Concentration gradient – different concentration in two areas ▪ Determines direction of movement of non-charged molecules ▪ Electrochemical gradient – charge + concentration gradient Na+ ▪ Determines direction of movement of charged molecules ▪ Membrane potential voltage difference across the membrane (imbalance of electrical net charges) *Arrow thickness represents rate of transport  6 1.B. Transport mechanisms PASSIVE No energy ▪ Direction of transport → down concentration or electrochemical gradient requirement a) Simple diffusion b) Facilitated ▪ Channel – hydrophilic pore ▪ Transporter/Carrier mediated – binds solute ACTIVE Energy ▪ Direction of transport → against concentration or electrochemical gradient requirement a) Primary – ATP hydrolysis b) Secondary  7 2.1. Passive Transport – Simple Diffusion ▪ No membrane protein required for transport Permeable Impermeable Molecules: ▪ Small nonpolar – CO2, O2 ▪ Small polar molecules – ethanol, water ▪ Large nonpolar – oils, steroids Diffusion rate: ▪ Permeability coefficient ▪ Difference in concentration gradient Rate of diffusion [Solute to be transported]  8 2.2. Passive Transport – Facilitated Diffusion ▪ Multi-pass membrane protein required Protein: for transport ▪ Channels – hydrophilic pore Molecules: ▪ Charged ions – K+, H+, Cl-, Ca2+, Na+ ▪ Polar molecules – glucose, amino acids ▪ Transporters/carriers – solute binds to ▪ Charged ion – K+, H+, Cl-, Na+ transporter → conformational change → solute released on the other side Hydrophilic pore  9 2.2. Passive Transport – Facilitated Diffusion A. TRANSPORTERS AND CARRIERS Specificity: each transporter/carriers transport one molecule ▪ Specificity of carrier protein determined by amino acid sequence ▪ Passive – uniport ▪ Active – symport and cotransport Transport mechanisms – alternating conformational model ▪ Solute binds to transporter → conformational change → solute released on the other side ▪ One molecule at a time ▪ Direction of transport determined by concentration gradient  10 2.2. Passive Transport – Facilitated Diffusion A. TRANSPORTERS AND CARRIERS Saturation (all carrier proteins Transportation rate: are occupied) Rate of diffusion ▪ Concentration gradient Vmax ▪ Affinity of the carrier protein for the solute ▪ Carrier protein availability ▪ Only ‘so many’ molecules can be transported [Solute to be transported] at any given time Example – Glucose absorption by erythrocytes P Glucose-6-phosphate  11 2.2. Passive Transport – Facilitated Diffusion B. ION CHANNELS ▪ Multi-pass channel membrane protein required for transport Specificity: Na+ channel ▪ Central region of channel – selectivity filter ▪ Discriminate based on charge and size Transport mechanism: ▪ Hydrophilic pore that allows passing of ions ▪ Direction of transport determined by electrochemical gradient + K channel  12 2.2. Passive Transport – Facilitated Diffusion Vmax B. ION CHANNELS Types: Saturation Rate of diffusion (all channel ▪ Non-gate controlled (K+ leak channel) proteins are open) ▪ Gated (open and closed conformation) ▪ Voltage-gated [Solute to be transported] ▪ Ligand-gated ▪ Mechanically gated Transportation rate: ▪ Electrochemical gradient ▪ Ion channel conductivity – how fast can ions pass through the channel ▪ Open conformation of channel  13 2.2. Passive Transport – Facilitated Diffusion B. ION CHANNELS ▪ Voltage-gated channel - open and close in response to changes in the membrane potential ▪ Refractory period – period of time that prevent re-opening of channels ▪ Guarantees directionality of signal propagation ▪ Example: sodium channels in action potential – channel opens when membrane depolarizes  14 2.2. Passive Transport – Facilitated Diffusion B. ION CHANNELS ▪ Ligand-gated channel – ligand binding triggers opening of the channel ▪ Ligand is not transported! ▪ Intracellular ligand or extracellular ligand ▪ Example: nicotinic acetylcholine receptors ▪ Chemical signal → electrical signal  15 2.2. Passive Transport – Facilitated Diffusion B. ION CHANNELS ▪ Mechanically-gated channel - respond to mechanical forces that act on the membrane (e.g. waves) ▪ Example – auditory hair cells ▪ Silence – channels are closed ▪ Sound waves create vibrations that deflect the stereocilia on hair cells ▪ Open of mechanically-gated ions → depolarization and transmission of signal  16 3. Active transport Energy Direction of transport – against concentration or electrochemical gradient requirement 1. Primary active transport – maintain ionic gradients across membranes ▪ ATP powered pumps – ATP hydrolysis expense driven transport 2. Secondary active transport: ▪ Coupled transporters (transporter/carrier proteins) – symporter & antiporter ▪ Cotransport – gradient driven transport 3. Light driven pumps – bacteriorhodopsin Light driven pumps  17 3.1. Primary Active Transport - pumps ▪ Pumps – transmembrane proteins (some are multi subunit) + ATP hydrolysis P-type pump – H+, Na+, K+, Ca2+ pump ATP hydrolysis → phosphorylation of pump Location: ▪ Cell membrane of plants, fungi, bacteria (H+ pump) ▪ Cell membrane of higher eukaryotes (Na+/K+ pump) ▪ Cell membrane of eukaryotes (Ca2+ pump) ▪ Cell membrane of sarcoplasmic reticulum (Ca2+ pump) V-type proton pump – H+ pump Pump is not phosphorylated Location: ▪ Plasma membrane of osteoclasts and some kidney tubule cells ▪ Vacuolar membrane of plants and fungi ▪ Endosome/Lysosomes membranes  18 3.1. Primary Active Transport - pumps F-type ATPase (ATP synthase) – H+ pump Structure: ▪ F0 – transmembrane pore F0 ▪ F1 – ATP binding site Function: F1 ▪ Normal direction – H+ pump ▪ Reverse direction – ATP synthesis (ADP + Pi → ATP) Location: ▪ Cell membrane of bacteria ▪ Inner mitochondrial membrane (all eukaryotes) ▪ Thylakoid membranes (plants)  19 3.1. Primary Active Transport - pumps ABC transporter (ATP binding cassette) – ions and small molecules ▪ ABC protein is specific for a single substrate ▪ ATP binding and hydrolysis drives transport Structure: ▪ Two transmembrane domains ▪ Two cytosolic ATP-binding domains Location: ▪ Bacterial plasma membranes (amino acid, sugar, and peptide transporters) ▪ Mammalian plasma membranes (transporters of phospholipids, small lipophilic drugs, cholesterol, other small molecules)  20 3.1. Primary Active Transport - examples P-type ATPase – Na+/K+ Pump ▪ ATP hydrolysis ▪ 3x Na+ cytosol – extracellular ▪ 2x K+ extracellular – intracellular Function ▪ Generation and maintenance of resting membrane potential ▪ Na+ (higher extracellular) ▪ K+ (higher cytosol) ▪ Regulate osmotic pressure ▪ Secondary active transport  21 3.1. Primary Active Transport - examples P-type ATPase – Na+/K+ Pump ▪ Cyclic mode of operation of the Na+/K+ pump  22 3.1. Primary Active Transport - examples P-type ATPase – Ca2+ Pump ▪ ATP hydrolysis ▪ Import 2x Ca2+ from cytoplasm into lumen sarcoplasmic reticulum ▪ Sarcoplasmic reticulum (specialized ER in muscle cells) Function: ▪ Maintain intracellular Ca2+ levels at minimum ▪ Calcium – primarily involved in cell signaling  23 3.1. Primary Active Transport - examples P and V pumps – H+ pumps P-pump V-pump V-pump  24 3.1. Primary Active Transport - examples ABC-type – CFTR Cl- channel ▪ Cell membrane of epithelial cells (airways, intestines) Healthy individual ▪ CFTR exports chloride ions → followed by water ▪ Maintains hydrated mucus Cystic Fibrosis – disease caused by H2O dysfunctional CFTR ▪ Symptoms: reduced chloride ion transport ▪ This disrupts the balance of salt and water in various tissues CFTR ▪ Thick Mucus Production ▪ Recurrent respiratory infections CFTR – Cystic Fibrosis Transmembrane Conductance Regulator  25 3.2. Secondary Active Transport - cotransport ▪ Coupled transporters (transporter/carrier proteins) – symporter & antiporter ▪ Cotransport – gradient driven transport ▪ Animal cells – Na+ driven cotransport ▪ Na+/K+ pump generated Na+ gradient ▪ Plant cells – H+ driven cotransport ▪ H+ pump generates H+ gradient  26 3.2. Secondary Active Transport - examples Symport Na+-glucose ▪ Active transport of glucose coupled to Na+ electrochemical gradient  27 3.2. Secondary Active Transport - examples Co-transport examples Antiport Na+/Ca2+ Ca2+ Assists in maintaining Symport Ca2+ Glucose Na+/Glucose low intracellular Ca2+ 3 Na+ 2 Na+ Glucose Cytosol Na+ ADP+Pi 2 K+ H+ Antiport ATP Na+/H+ Na+/K+ ATPase (pump) Helps regulate H+ Na+ intracellular pH 3 Na+  28 4. Action potential and synapse ▪ Each cell – own set of membrane transport ▪ Specific functions ▪ Multiple membrane proteins function together  29 4. Action potential and synapse MEMBRANE POTENTIAL ▪ Distribution of ions on either side of the lipid bilayer gives rise to the membrane potential ▪ K+ leak channels – maintain resting membrane potential (-60mV)  30 4. Action potential and synapse ▪ Messages are carried by action potentials ▪ Mediated by voltage-gated cation channels (Na+ and K+)  31 4. Action potential and synapse ACTION POTENTIAL ▪ Triggered by depolarization of nerve cell plasma membrane  32 4. Action potential and synapse ▪ Propagation of action potential  33 4. Action potential and synapse Nerve terminal - synapse ▪ Electrical signal is converted into a chemical signal ▪ Arriving action potential – Ca2+ voltage-gated channels – influx of Ca2+ stimulates exocytosis of synaptic vesicles (neurotransmitter)  34 Postsynaptic cell POSTSYNAPTIC CELL ▪ Neurotransmitter binds to a ligand-gated ion channel ▪ Chemical signal converted into electrical signal ▪ Example: neuromuscular junction ▪ Excited motor neuron releases acetylcholine → opens ligand-gated Na+ -> influx of Na+ → depolarization → voltage-gated Ca2+ channels open → Ca2+ in sarcoplasmic reticulum open too ▪ ↑↑↑↑↑Intracellular Ca2+ → muscle contraction