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Cellular Physiology – Dr. Donglin Bai.docx

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Western University
Physiology 3140A
Donglin Bai

Cellular Physiology – Dr. Donglin Bai Introduction – Electric Currents for Communications • Electric currents are used by cells in the brain & muscles for cellular communication • Diagnostic tests of heart & brain detect electric currents & physiological measurements can be made in single cells using electric recordings • Main proponent of electricity from metals was Volta – volts • Main proponent of animal electricity was Galvani • Galvani used natural electricity (lightning), knife (metallic objects), & touching of cut nerve to its uncut surface to elicit contraction • Contemporary volta interpreted it as electricity generated by bimetallic junction (battery) • Electrical currents are used by brain cells & muscles for generating signals for communication • Electrocardiogram o Measure electric currents from the heart • Electroencephalogram o Measures brain electrical activities from different electrodes placed on scalp Term Unit Typical Range in Cellular Physiology Potential Volt (V) mV (10 V) μV (10 V) -9 -12 Current Ampere (A) nA (10 6) pA (10 9A) Resistance Ohms (Ω) MΩ (10 Ω) GΩ (10 Ω) Conductance Siemens (S) nS (10 S) pS (10 -1S) mV = millivolt nA = nanoAmpere MΩ = megaOhm nS = nanoSiemens pA = picoAmpere GΩ = gigaOhm Ps = picoSiemens • • Direction of electric current – movement of positive charges • Positive charges moves from a point of high (electric) potential to a point of low potential • Larger the potential (voltage) difference, the larger the current • Current depends on the medium that carries the current • Small diameter cable offers high resistance than a large-diameter cable • Analogy: Ball rolling down a hill  Vertical height = potential  Speed of ball = current  Surface the ball rolls on = resistance o Steeper the hill the lower the resistance & faster the ball will move • Pressure & water flow are analogous to voltage & current respectively • In excitable cells – current is carried by ions (versus electrons in metals) • Ions = cations (+ve) & anions (-ve) o Cations: Na , K , Ca , Mg 2+ o Anions: Cl & HCO 3- Ohms Law • Applies to electric circuits • Current (I) passing through a conductor is directly proportional to the voltage difference (V) across the conductor & inversely proportional to the resistance (R) of the conductor Ion Channels • Control resting (passive) & active properties of the excitable membrane • Excitable membranes are those that have action potentials or other voltage-dependent ion channels • All excitable cells (all able to fire AP) - have ion channels on cell surface membranes • Important ions: Na , K , Ca & Cl- • Components of Ion Channels o Pore – allowing ion conduction o Selectivity filter – determining ion selectivity (how we categorize channels) o Gate – regulating opening & closing, controlled by sensor o Sensor – channels utilize several forms of sensors • Working Hypothesis of Channel o Shown as transmembrane structure with central pore region that spans the membrane o Allows charged ions to transverse the generally impermeable lipid bilayer • Sensor – senses environmental changes & controls opening & closing of the ion channel • Opening and closing of an ion channel is known gating Types of Sensors • Electrical (voltage-gated ion channels) o Most Na channels o L- & T-Type Ca channels • Chemical (ligand-gated channels) o Fastest o Ligand changes conformation of channel o Neurotransmitter o nAchR (nicotinic acetylcholine receptor) o AMPAR (AMPA receptor) o NMDAR (N-methyl-D-aspartate receptor) o GABA RA(γ-aminobutyric acid Aeceptor) • Mechanical (mechanically gated ion channels) o Pressure o Temperature • **G-protein coupled receptors are the slowest **not all ligand-gated channels have 2 binding sites + Voltage Gating: Na Channel Activation • Physical movement of the sensory is detected for voltage gating of Na channel responsible for ap • Gating of Channels o Channels exist in either the closed or open configuration o Subtle changes in conformation underlie opening of a channel o When there are mutations – sensor changes gate subtly but current can still be recorded Physical Properties of Ions • Selectivity of ion channels is based on: size of ions & their charges • Charge density: o Size & charge Very subtle change in of an close configurations ion o Increases with increase of ion charge & reduction in ion size o Parameter critical for hydration levels – determine how many water molecules are going to be stuck with the ion o Average number of water molecules stuck in a hydration number o Larger the hydration number – harder is for a hydrated ion to move • Mobility – measured by velocity of the ions (in μm/s) per electrical field strength (V/cm) • Atomic weight is not directly related to the hydration number Hydration of Ions + + Potassium (K ) Sodium (Na ) Size (radius) 1.33 A 0.95 A Charge + 1 + 1 Hydration # 2.9 4.5 Physical Na has an increased Hydration number than K+ Properties of Na ,+ K & Cl - Ion Atomic Number Crystal Radius (A) Hydration Number Mobility (μm/s)/(V/cm) Na + 23 0.95 4.5 5.2 K + 39 1.33 2.9 7.64 - Cl 35.5 1.81 2.9 7.91 • Hydration depends on charge & size (or charge density: amount of charge per unit volume) • Hydration number determines ionic mobility in water – higher hydration # = decreased mobility Components of Ion Channels • -ve charged amino acids are concentrated at cytosolic entrance to pore & attract cations & repel anions o Channel cation-selective • Portion of channel polypeptides (selectivity loop) forms a selectivity filter: short, rigid, narrow pore, lined by carbonyl oxygen atoms of their polypeptide backbones • Size of this narrow pore determines selectivity for bacterial K+ channel Hydration of Ions & Interactions of Ions with Selectivity Filter • Water acts as a dipole to stabilize ions • Charged residues in region of selectivity filter (very rigid) replace water & stabilize each ion as it passes through the pore  energy change minimal • K -selective filter is optimal for K but not for the smaller Na • Summary o Ion channels are membrane proteins that form pore structures across cell membrane o It opens/closes in different conditions, which allows/stops flow of certain ions o Electrical activity of neurons (excitatory and inhibitory) is almost entirely through opening & closing of ion channels Water is removed from ions by carbonyl O Molecular Structure of Ion Channels Number of Subunits or Domains • Different number of subunits (or domains) is found in different group of ion channels • Exceptions exist • Increase in number of subunits/domains = larger pore space Oligomeric Channel Proteins • Homo = same (subunit) • Hetero = different (subunits) • Oligo = more than one e.g. dimer, trimer, tetramer Nicotinic Acetylcholine Receptor (nAChRs) • Ligand-gated ion channels at neuromuscular junction • Junction (synapse) between a nerve & skeletal muscle & also present in the brain • Pentameric – 2 α, 1 β, 1 γ, 1 δ • Each subunit shares similar topology o All have 4 transmembrane domains o TM2 – M2 (transmembrane domain - contributes the pore lining) • Receptor (binding to Ach) is the same molecule as the ion channel • Once Ach binds to receptor (on 2 α subunits) • Channel opens by a 15 degree rotation of the M2 residue (very subtle) – 3 Angstroms wider • Selective for cations (nonselective cation channels) + + 2+ o Na & K ; also Ca for some types o Residues on TM2 are negative – repelling anions + Voltage-Gated K Channel • Tetramers (different subunits) • Each subunit is composed of 6 transmembrane domains o With amino- & carboxy-terminal regions inside the cell (intracellular) • S4 segment is full of +ve charges & represents the voltage-sensor region (arg) • Pore loop (H5) contains the selectivity filter – site for binding of various channel blockers (Ex. tetraethylammonium – TEA) • Narrow part of channel closes • Ion selectivity o Carbonyl oxygens on peptide chain in the region of the selectivity filter replace water and stabilize each ion as it passes through the pore o K+ selective filter – optimal for K+ not Na+ • Structure of a K+ channel voltage sensors o Found that K+ channel voltage sensors resemble charged ‘paddles’ o S4 voltage sensing regions contain + charged arginines o Hinged gates - allow channel pore to open or close o RMP – Resting state  Outside is positive & inside is negative  Gray +ve charge - S4 area  S4 is very close to the cell interior  Channel pore opening at cytoplasmic face is closed o Membrane depolarization   Remove charges & reverse charge  Inside more +ve  S4 region moves up  Changes channel conformation - gates open o Selectivity filter never changes o Wider part of channel is closing (bottom) 2+ + Voltage Gated Ca & Na Channels • Only one protein subunit (very long) to form pore • Each subunit is made of 4 domains (I – IV) • Each domain has 6 membrane spanning segments • Only 1 subunit is enough to form channel (unlike K which requires 4) • Voltage-gated channels o Some channels have two gates:  Activation gate  Inactivation gate o During resting state  Outside +ve  Inside –ve  Activation gate is closed (only need this channel to close to make pore not penetrable) o Membrane depolarization (very strong)  Inside +ve  Outside –ve  Activation gate opens  Inactivation gate is open  Channel is conducting o After a little time, inactivation gate closes due to significant depolarization o Both gates in closed state – reversing back to RMP  Inactivation gates closes & opens very closely • Inactivation of Voltage-Gated K+ Channel (A-type K+ channel) o For this channel the inactivation depends on amino terminus  Remove 20 amino acids at N-terminus: no inactivation  Restore 20 amino acids: restore inactivation • Ball & Chain Model of Channel Inactivation o Depolarization leads to channel activation, followed by a delay with blocking of pore by N-terminus (inactivation) o Membrane hyperpolarization is required for the removal of inactivation Gap Junctions • Couple neighboring cells together by creating a pore from the cytoplasm of one cell to the cytoplasm of another • Proteins that span the membrane of one cell • Each half of a channel, connexon, or gap junction hemichannel – is made of 6 protein subunits called connexins • Each connexin has 4 transmembrane domains 1 connexon (half-channel) = 6 connexins = 6 x (4 transmembrane domains) • Amino terminal & carboxy terminal inside the cell • Homomeric & homotypic = both the same channel & both the same constituents within each channel • Homomeric & heterotypic = same constituents within each channel but different half channels • Heteromeric and heterotypic = different constituents within each channel & different half channels • When 2 opposing connexons from neighbouring cells are docked together – gap junction forms • Responsible for cell-to-cell communication in many cell types o Neurons (electrical synapse) o Cardiac cells (constituent of intercalated discs – junction between 2 cardiomyocytes) o Smooth muscle cells • Properties o Provide cytoplasmic continuity between cells – current passes between cells typically in both directions (electrical synapse) – easier to go one way for some junctions o Electrical synapse has almost no synaptic delay (0.1 ms) ** important for cardiac function o Synchronous activation of a large number of cells in heart & smooth muscles o Channels are large enough to pass small molecules (<2+kD) (nutrients & signaling molecules) o Increased intracellular acidity (decreased pH) and/or Ca close gap junction channels Methods of Measurements Summary of Conventions & Diagram Symbols Electricity in the body is minimal – need a very good amplifier Anything with potential is considered a battery # of open/closed ion channels - resistance - Adjustable • Voltage (V) o Potential o Units: volts o In physiology we usually encounter millivolts or microvolts (10 ) • Charge (Q) o Similar charges repel – for a battery anode attract anions & cations are attracted to cathode o Units: Coulombs (C) • Current (I) o Rate of movement of charge (dQ/dt) o Units: amperes (1 C/s = 1 A) o In physiology we usually encounter nanoamperes (nA – 10 ) or picoamperes (pA – 10 ) o Movement of positive charges (or positive ions) • Ohms Law o Voltage difference between two points is V o Current (I) flows between two points o Resistance is defined by: V = I R o Units: Ohms • Conductance o Permeability of the membrane to an ion o Inverse of resistance G = 1/R o Units: Siemens (S) o In physiology we usually encounter one to a hundred picosiemens (10 ) Ohms Law I = V/R = GV • All potentials or physiological signals have be made with respect to a reference location • For intracellular, or patch electrode recordings – extracellular medium is where reference electrode is placed & defined as 0 mV • Electrical activity can be measured by two main types of electrodes – sharp or patch electrodes • Signal measured can be voltage (using current clamp mode) or current (using voltage clamp) Measure current at clamped (fixed) voltage: voltage clamp Measure voltage at clamped (fixed) current: current clamp (imposed current is 0 or constant) Sharp Electrode • Glass pipette pulled to fine taper (so as not to destroy the cell but resistance is high) (<0.5 μm diameter) – filled with conductive electrolyte & penetrates the cell • High resistance electrodes (high noise) • Single channel activity buried in noise, not distinguishable • Records intracellular voltage (I-clamp) • Membrane seals around the electrode to provide stability of recording • Adjusts amplifier to 0 for reference point • Sudden drop (-65 mv) is usually RMP • Current clamp mode • Advantages o Simple direct measurement of voltage as function of time o Emulate in vivo conditions in the muscle & brain • Limitations o Limited control of membrane potential (hard to inject current) o High resistance electrode gives more noise (many channels on membrane - hard to separate 1) Voltage Clamp versus Current Clamp • Conventional sharp-electrode intracellular recordings – membrane potential (voltage) is recorded • Similarly voltage recordings can made by whole-cell patch recording • The recording of membrane potential is sometimes referred to as current clamp – since the net membrane current is zero • Current clamp recordings (potential versus time records) are similar to what happens in physiology but is difficult to separate different potentials or channels • Alternative is to study current under fixed voltage – voltage clamped o Specialized electronic equipment is required Voltage-Clamp • 2 electrodes o One recording cell membrane potential o Other injecting current • Cell follows voltage command • Voltage is clamped at desired potential • Advantages o Voltage is under control – important since many ionic channels are voltage dependent o Quantitative measure of current or conductance G = 1/V at a fixed voltage o Current is reflecting conductance – channels open or closed I = V x G therefore I α G (conductance – how many channels in open state) • Limitations o Technically demanding Voltage Clamp: hold the cell at -70 mV  jump to -10 mV Patch-Clamp Recordings • Use low resistance pipettes • Relatively larger tips (1 μm) than sharp electrode • Gentle suction through patch pipette – main technique to form a tight seal with the membrane • Recordings can be obtained from a small piece of membrane & whole cell • Can record channel activity of suctioned channels on small portion of the membrane • Suction is gentle • Can look at intracellular components • Don’t break membrane • Provide Ach to cell  record single-channel activities using neurotransmitter 4 Configurations of Patch Recordings • Whole cell recording- gives voltage & current of whole cell • Other patch configurations are used to record single channel currents & can be manipulated by adding drugs (ex. neurotransmitter) • Different chemicals can be applied in bath to modulate the ion channels on the inside of the membrane in the inside-out patch Outside-Out Patch • Eventually membrane will fuse back together • Used to determine which ion channel you are using Advantages • Good for I-clamp • Good for V-clamp • Able to measure single channel activities • High signal to noise ratio (one electrode) Limitations • Washout of cell content in certain configurations I-clamp versus V-clamp • • Current clamp • Voltage clamp o V (commonly in mV) o I (commonly in pA, nA) o Sharp electrode o Two-electrode V-clamp o Patch clamp (whole cell – don’t see o Patch clamp (all recording modes) single channel) Patch-Clamp Recording • Single-channel currents can only be studied using low- resistance patch electrodes (& not sharp electrodes) • Single channel current/amplitude is constant (at fixed voltage) • Random appearance of channel openings & closings • Durations of opening is variable – dealing with probabilistic process • Current transitions are essentially instantaneous – very little time required for transition o When held at membrane potential of 0 mV, unitary currents are 10 pA (10amperes) o Corresponds to roughly to 100,000,000 K ions per second traveling through channel Ionic Distribution & Nernst Equation • Ionic concentration differences exist across cell membrane • If concentration was the only force – ions would readily diffuse through pores & channels – concentration gradient destroyed • Concentration gradients are maintained by: o Membrane is not permeable to all ions equally o Electrical gradients – illustrated at equilibrium by Nernst Equation o Ionic pumps • As a result of ion movements – electrical potential difference or membrane potential exists • Potential difference (mV) can be found across all membranes of living cells & organelles • Separation of charges: gives rise to potential or voltage gradient • Similar charges repel & opposite charges attract • Diffusion coefficient (D): describe how fast an ion (or atom) can diffuse through a membrane barrier • Forces on an ion o o o o o o o o o Chemical force: depends on concentration gradient & absolute temperature (T: 273 + °C – measured in K) o Electrical force: depends on charge & electrical gradient (potential) o Total force: chemical force + electrical force • Diffusion of Electrolytes (charged solutes) o 2 forces: diffusion (concentration force) & electrical force (electrical field or voltage gradient) o Diffusion  Passive – no energy requirement  All particles including ions & electrolytes diffuse from high to low concentration  Chemical force – proportional to concentration gradient & diffusion constant specific for that molecule  Depends on concentration gradient & absolute temperature o Charged particles experience electrical force  Electrical force = charge x electrical field • Convention for electrical field is the same as current – positive charge travels along electrical field & negative travels in opposite direction • Total force = chemical force + electrical force will determine ion movement Assume, K moves faster than Ac- because it is +vely charged – moves down its [ ] gradient from 1 to 2 D K+ > D Ac- Transient charge separation at membrane: excess +ve charge in 2 & excess –ve charge in 1 Electrical field points from compartment 2  1 Electrical field arising from diffusion of ion Voltage gradient or electrical potential difference: diffusion potential Arises because K+ moves faster than Ac- through pores Electrical field or diffusion potential will: • Speed up movement of slower ion (Ac-) to 2 • Slow down the faster K+ ion **resistance of more +ve charge flow After a long duration: Ac- will catch up with K+ – diffusion potential = 0 No excess charge + or - are found in compartments Wh+n K -c-1= KAc-2 charge neutrality in each compartment K 1 Ac 1 K 2 Ac -2 Example 2: • No Ac- movement across membrane • Compartment 2 will remain +ve in potential with accumulation of +ve charges that repel further K+ movement • Same initial conditions as ex. 1 but Ac- cannot follow K+ across because pore is not permeable • K+ will flow down concentration gradient from compartment 1 to 2 until the electrical force produced by movement of K+ ions is sufficiently large to prevent further net movement of K+ • Electrical equilibrium potential (m ) is calculated from Nernst equation – defined as potential at which the chemical (concentration) force is exactly balanced by electrical force acting on the ion • Equilibrium – movement of ions continues but movement in one direction is balanced When will diffusion of K+ stop? • Diffusion of K+ will stop when the chemical force is balanced by an electrical force resulting in electrochemical equilibrium (ion distribution is still unequal) Nernst Equation At 27° the equation is E = 60 log [K] 2 in mV K [K] 1 No equilibrium potential for Ac- since this ion cannot diffuse through membrane Considering a membrane permeable to ion X with an uneven distribution in & out of a biological cell Nernst equation becomes: Ion Distribution in Living Systems: Mammalian Muscle Na & K are somewhat opposite Highest difference Proton concentration slightly different Cell membrane is balanced • Cells must contain equivalent amounts of +ve & -ve charges, that is, be neutral • Therefore, many other anions, in addition to Cl-, must be present • Many cellular constituents (proteins, nucleic acids) are negatively charged, and comprise the other –ve charges need to neutralized K+ Equilibrium Potentials for Each Ion • Dependent on ionic distribution, each cell type has its own equilibrium potential for each ion species • Common values: o E K+-89 approx -100 mV o E Na+-45 approx. +60 mV o E Cl-65 approx. -75 mV How is polarity of EKdetermined? Electrical gradient gradually builds up with more & more K+ ions move out of cell generating electrical gradient Electrical gradient generates electrical force that eventually balances chemical forces to reach equilibrium Equilibrium potential for K+ ions in muscle is -89 mV Note: RT/F * 2.3 = 60 mV at 27°C Equilibrium potential for each ion: • Dependent on ionic distribution, each cell type has its own equilibrium potential for each ion species • Calculate equilibrium potentials (ENa+& E Cl-or mammalian muscle cell Resting Membrane Potential • Cell resting membrane is important parameter for excitable cell • Equilibrium potential & membrane potential determine direction & magnitude of ion movement (current) Current-Voltage Relationship • Amplitude divided by potential = slope (conductance) • Many ion channels have a curved graph – rare to find a linear graph • Two important characteristics o Reversal potential: membrane potential at which current is reversed o Conductance: slope of the curve (increase in conductance = steeper slope) G = I / V Current-Voltage Relationship (IV) & Cell Membrane Potential (E ) m • Predict driving force & current flow direction If we use K+ ion as an example: IK= G KE m E ) K E m -65 mV E K -89 mV IK= G K - 65 – (-89)] = 24 G K When E =m-100 mV, I = -K1 G K At membrane potential of -65 mV, K current (I ) Ks outward current At membrane potential of -100 mV, K current (I ) Ks inward current Calculate Na ions: INa G (Na– m ) Na At membrane potential of -65 mV: E Na+60 mV: I = Na(E –Na )m= -1Na G Na - + + Cl, K , Na - dominate ions that determine cell membrane potential When >1 ion determines membrane potential - use Goldman-Hodgkin-Katz Equation (Goldman Equation) • Nernst equation gives equilibrium potential for a single ion • Goldman equation gives equilibrium potential for multiple ions Resting membrane potential approx. -70mV for many neurons – determined to a degree by ions Na+ & Cl- Nernst equation: Goldman equation: ** intracellular chloride is on top Where: E m membrane potential K o
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