BM Voltage Gated Ion Channels PDF

Fundamentals of Membrane Potential You will be able to Discuss the meaning of the term ‘Equilibrium Potential’ Appreciate how changes in ion fluxes can be exploited by the cell to effect cellular events Discuss the basis for the establishment of the RMP Understand how the patch clamp apparatus...

Fundamentals of Membrane Potential You will be able to Discuss the meaning of the term ‘Equilibrium Potential’ Appreciate how changes in ion fluxes can be exploited by the cell to effect cellular events Discuss the basis for the establishment of the RMP Understand how the patch clamp apparatus in conjunction with expression of voltage gated channel genes may be used to learn about channel physiology Understand the structural basis for ion selectivity, voltage gating, and channel inactivation exhibited by many voltage-activated channels Appreciate the importance of these structural features in neural transmission The Movement of Ions across Membranes  Diffusion  Dissolved ions distribute evenly  Ions flow down concentration gradient when:  Channels permeable to specific ions  Concentration gradient across the membrane  Unequal movement of ions will cause an alteration in the distribution of charges across the membrane  Potassium Equilibrium Potential = Membrane potential difference at which movement down concentration gradient equals movement down electrical gradient; electrical gradient equal to and  Potassium Equilibrium Potential For a typical neuron  Extracellular K+ ion concentration = 5 mM  Intracellular K+ ion concentration= 100 mM  The RMP = -70 mV  The concentration gradient on its own would generate an efflux  The electrical gradient on its own would generate an influx  An efflux is what actually happens. Thus the concentration gradient under these conditions prevails  The equilibrium potential – when the two forces are equal (and there is no net movement of K+ ions) is given by the Nernst equation  Potassium Equilibrium 100 mM Potential 5 mM Outward chemical gradient K+ Ek = -78 mV - + Inward electrical gradient At -70 mV (for a normal resting cell), the outward chemical gradient > inward electrical gradient hence K+ current is outwards BUT AT -85 mV (if EK is -80 mV), the inward electrical gradient > outward chemical gradient K+ CURRENT - 65 mV - 85 mV - 105 mV  Potassium Equilibrium  As predicted Potential  Membrane potential is sensitive to extracellular K+  Increased extracellular K+ depolarizes membrane potential and can cause cells to be abnormally excitable, may generate spurious action potentials  It needs to be regulated  Regulating the External Potassium Concentration in CNS  Blood-Brain barrier  Potassium spatial buffering  How does the RMP come about?  Most mammalian cells have RMPs more positive (-70 to - 40 mV) than EK as there is always some leakage pathway(s) for Na+ & sometimes Cl– ie permeability of K > Na, > Cl at rest Consequently the finite permeability of the membrane to Na+ (and Cl–) prevents the RMP from actually reaching the Nernstian K+ potential  Thus the extent to which each ion gradient influences the membrane potential is defined by the permeability of the membrane to that ion. i.e. Even with a large concentration gradient, an ion may exert little influence if the Pi value is small (Note: Large changes in Vm, minuscule changes in ionic concentrations) P K [K + ]in + P Na [Na+ ]in + P Cl [Cl - ]out Vm  -60 log + PK[K ]out + PNa[Na+]out + PCl [Cl-]in This influence of multiple ions is reflected in the  Why is this important?  It is the rapid large changes in the ratios of permeability for the different ions that is the basis of bioelectric phenomena For instance the large increase in PNa (via V-gated sodium channels) is the basis of the transient depolarization of the membrane potential associated with the neuronal action potential and similar alterations in permeability (via activation of channels) is the basis of sub and suprathreshold potentials (ie graded and action potentials) For example, during rest in the squid giant axon the permeabilities have the ratio PK:PNa:PCl = 1: 0.03: 0.1 so that Vm = -70 mV but during the depolarizing phase of an action potential the ratio is PK:PNa:PCl = 1: 15: 0.1 so that Vm = +44 mV Physiology of Voltage-gated ion channels  Methods for recording ionic currents Two electrode voltage clamp Vout V I Vin Cell Iout Vin - the command voltage Vout - the measured voltage Iout - the measured current  Methods for recording ionic currents atch clamp configurations Suction Cell-attached (giga-ohm seal) Pull More suction (low Ca2+) Pull Inside-out Whole-cell Outside-out patch recording patch  Properties of ion channels - Selectivity  As observed for the action potential, ion channels are very permeable to some ions but not others. Hence Na+ channels allow Na+ ions to permeate much more readily than K+. K+ channels exhibit converse behaviour.  This is known as the channel SELECTIVITY for ions  Clearly ion channels do not exist in the conducting (open) state all the time as this would quickly dissipate the electrochemical gradient and kill the cell So permeation must be brief and controlled Hence GATING and INACTIVATION  Properties of ion channels - Gating Most channels spend the vast majority of time in the non- conducting (closed) state and require some energy input to elicit opening. The process by which a channel opens and closes is known as channel GATING, and is reversible. Gating is not coupled to the i.e. C O movement of ions through the pore of the open channel Minimal energetic interaction between the transported ion and channel Ion flux is limited by the status of the channel (open or closed) Gating regulated by protein conformation and is independent of permeability control  Properties of ion channels -  The majority of ion Gating channels are often described in terms of the factor that controls the gating process: Voltage-gated channels e.g. Na+, Ca2+ and K+ Ligand-gated channels e.g. Ach, GABA  However the gating process itself may be altered by a secondary factor, a process often termed channel MODULATION - e.g. by altering the rate of channel gating or the membrane potential at which gating occurs (may be driven by second messengers or enzymatic processes in cells)  Modulation may last for seconds - minutes - hours  When ion channels open they conduct the permeating ion(s), but other ions or foreign molecules also try to pass through. Certain ones will enter pore but not permeate and these cause ion channel  Properties of ion channels - Inactivation  A change in the membrane potential is required to gate such channels from a closed or RESTING state to a conducting, or OPEN state. driven by depolarisation C O  Once voltage-gated channel is in the open state there are two possible routes to a non-conducting state again.  Channel can return to closed state a process termed DEACTIVATION Cdriven by repolarisation O  OR channel driven by maintained depolarisation to different non- conducting state termed INACTIVATION C Odriven by depolarisation I Structural features of Voltage-gated ion channels The structure of voltage- activated ion channels ( subunits) channel Sodium + + + + + + + + Potassium 2345 6 2345 6 2345 6 2345 6 + + + + channel + + + + + + I II III IV 2345 6 + + Calcium channel + + + + X4 + + + + 2345 6 2345 6 2345 6 2345 6 + + + + + + + + I II III IV Selectivity Filter : The pore Loop OUT + + + + + + + + 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 + + + + + + + + COOH I N I II III IV NH2 Na + II I II I I I II I I V V Selectivi I ty filter I Selectivity Filter : The pore Loop SELECTIVITY FILTER Ion selectivity for Kv determined by carbonyl backbone groups of the TVGYG motif in the P loop For NaV channels by DEKA side chains Ion Channel Structure - Ion selectivity  Gating – The Voltage Sensor The positively charged amino acids are responsive to an increase in intracellular membrane voltage The S4 gets repelled and corkscrews upwards to open the pore and gate the channel Thus voltage activation and gating  Voltage-activated sodium channels - Gating  Sodium channel activation is associated with the movement of charge within the structure of the channel itself and this occurs before ion movement…….known as the GATING current + + Na+ current is + Resting + preceded by --- + + gating-current S4 helix Pore + INa+ + + Depolarised + + + +++ Membrane potential Inactivation – The IFM motif for NaV channels  I II III IV Fast OUT inactivation of + + + + + + + + 2345 6 2345 6 2345 6 2345 6 Na+ channels is + + + + + + + + mediated by an IN COOH IM intracellular F ‘gate’ that binds NH2 to the Hydrophobic triad intracellular forms inactivation gate mouth of the pore - a tethered pore blocker IFM peptide occludes the Na+ channel pore W T Na Na + + II II 5 ms I III I III IV IV G MF I GG IFM QQQ G I M F 20 Activated Inactivated ms Synthetic peptides with IFM motif can restore fast inactivation to The End