BI 2004 Essential Animal Cell Biology

1. BI 2004 Essential Animal Cell Biology Cell membranes 4 Ion channels Dr Gordon McEwan Department of Biomedical Sciences

2. 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

3. acetylcholine binding site Na+ acetylcholine lipid bilayer Adapted from ECB Fig 12-18 cytosol gate closed open overall structure 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

4. glass microelectrode conducting fluid tight seal ion channel cell membrane cell-attached detached patch metal wire trace of ion channel currents constant voltage source glass microelectrode metal electrode Adapted from ECB Fig 12-20 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

5. 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)

6. state of channel: open open open closed closed closed Current (pA) Time (ms) Ion channel recording (contd) 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

7. ligand-gated voltage- gated stress- activated extracellular intracellular out +++ +++ in --- --- closed Adapted from ECB Fig 12-22 open out + + in - - mechanical  membrane potential molecule binds to channel 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

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

9. 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

10. 0 mV Basis of membrane potential cation channel 0 mV cations move through channels  non-zero membrane potential charges balanced Adapted from ECB Fig 12-25

11. 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

12. 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

13. 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