Essential Animal Cell Biology Department of Biomedical Sciences Cell membranes 4 Ion channels Dr Gordon McEwan Department of Biomedical Sciences

Ion channels Channel proteins form transmembrane aqueous pores which allow passive movement of small water-soluble molecules into or out of the cell or organelle Most channel proteins in plasma membrane form narrow pores which are only permeable to inorganic ions - ion channels Ion channels are highly selective for specific ions (eg Na+, K+, Ca2+, Cl-) Ion selectivity depends on: diameter - narrow channels can’t pass large ions shape - only single ions of correct species can access charge - distribution of charged amino acids in pore Passage of ions through narrowest portion of channel rate limiting  saturable transport

Ion channel gating Ion channels not continuously open Can switch between open and closed state by changing conformation Conformation change regulated by conditions inside and outside cell acetylcholine binding site lipid bilayer gate cytosol Na+ overall structure closed open Adapted from ECB Fig 12-18

Ion channel recording Ion movements across membrane can be detected by electrical measurements Technical advances now permit measurement of electrical current through single channel molecule - patch clamp recording glass microelectrode conducting fluid tight seal ion channel cell membrane cell-attached detached patch metal wire constant voltage source trace of ion channel currents metal electrode Adapted from ECB Fig 12-20

Ion channel recording (contd) Patch clamp technique permits recording from ion channels in large variety of cell types By changing ionic composition of bathing solution - can establish which ions go through channels Can “clamp” membrane potential at different voltages and establish effects on channel activity If patch is sufficiently small, only one channel molecule present; Can measure ion flow through single channel - 10-12 amps = picoamp (pA) Current randomly switches between two levels due to channel being either open or closed Regulation  channel being in open (or closed) state for a greater proportion of time (but still opening and closing at random)

Ion channel recording (contd) Current (pA) Time (ms) state of channel: closed open Adapted from ECB Fig 12-21 Na+ channel activated by acetylcholine   open state probability (Po) When acetylcholine absent channel spends most of time in closed state

Classes of ion channel Two distinct properties of ion channels: (1) ion selectivity - type of ions which can pass (2) gating - conditions which influence opening and closing closed open out in voltage- gated ligand-gated extracellular intracellular stress- activated  membrane potential +++ --- + - molecule binds to channel mechanical Adapted from ECB Fig 12-22

Stress-activated channel eg auditory hair cells in ear basilar membrane auditory nerve fibres stereo-cilia tectorial membrane hair cells supporting cell channel closed linking filament entry of positively charged ions open Bundle not tilted Bundle Adapted from ECB Fig 12-23 Sound vibrations cause basilar membrane to move up and down  stereocilia of hair cells tilt  stretching of linking filaments  opening of channels

Voltage-gated channels Voltage-gated channels play major role in propagating electrical signals in nerve and muscle cells Channels have special charged protein domains - voltage sensors - which are extremely sensitive to changes in membrane potential When potential changes beyond threshold voltage, channel switches from closed to open configuration Open state probability increases at threshold potential Q What controls membrane potential? A Opening and closing of ion channels  control loop: ion channels  membrane potential  ion channels Fundamental principal of electrical signaling in cells

Basis of membrane potential cation channel mV mV charges balanced cations move through channels  non-zero membrane potential Adapted from ECB Fig 12-25

Resting membrane potential Negative charge on intracellular organic anions balanced by K+ High intracellular [K+] generated by Na+-K+ ATPase Large K+ concentration gradient ([K+]i:[K+]o  30) Plasma membrane contains spontaneously active K+ channels  K+ move freely out of cell As K+ moves out of cell, leaves negative charge build up  opposes further K+ exit At equilibrium, electrical force balances concentration gradient and electrochemical gradient for K+ is zero (even though there is still a very substantial K+ concentration gradient) Resting membrane potential = flow of positive/negative ions across plasma membrane precisely balanced

Resting membrane potential Membrane potential measured as voltage difference across membrane For animal cells, resting membrane potential varies between -20 and -200 mV Negative value due to negativity of intracellular compartment compared to extracellular fluid Because K+ channels predominate in resting plasma membrane, resting membrane potential mainly due to K+ concentration gradient Nernst equation permits calculation of membrane potential (V): V = 62log10(Cout/Cin) where Cout and Cin are extracellular and intracellular ion concentrations of monovalent cation at 37°C

Changing the membrane potential Resting membrane potential determined by K+ permeability If Na+ channels open, because [Na+]out>>[Na+]in, Na+ will move into cell  membrane potential less negative New equilibrium potential established - compromise between negative K+ potential and positive Na+ potential Membrane potential determined by state of ion channels and transmembrane ion concentrations Because electrical changes much more rapid than ion concentration changes - ion channel activity most important in controlling membrane potential Voltage-gated ion channels control electrical signalling in nerve cells