Cellular Neuroscience (207) Ian Parker

1. Cellular Neuroscience (207)Ian Parker Lecture # 4 - Ion channels: electrophysiology http://parkerlab.bio.uci.edu

2. Single ion channels extracellular Cell membrane cytosol Molecular structure Physical structure Functional model Simplified model

3. Single channel kinetics Transitions from open to shut are instantaneous Mean open time is a fixed characteristic of the channel Mean closed time shortens with increasing stimulus (e.g. depolarization or agonist concentration) Single channel current depends on channel conductance and electrochemical gradient for ion flow Channel opening does not require energy source (ATP), so channel continues to work in isolated membrane patch. Energy for ion flow (current) comes from electrochemical gradient across membrane

4. How big are single channel currents? Amps (log scale) 100 W light bulb 1 10-3 (mA) calculator 10-6 (mA) action potential at node of Ranvier Limit of conventional Voltage clamp e.p.s.c. (current evoked by 1 vesicle of neurotransmitter) 10-9 (nA) Single channel currents 10-12 (pA) One ion per ms 10-15 (fA)

5. Single channel current and conductance • Because single channel current varies with membrane potential and ion gradient, a better measure is the conductance of the channel (g). This is a fixed characteristic (‘fingerprint’) of a given channel. • g = i / (V-Veq) • (v = membrane potential : Veq = reversal potential for current flow through channel) • Unit of conductance is the Siemen (S : 1/Ohm) : single channel conductances are expressed in pS Single channel current (pA) 1 Line in red shows the current/voltage Relationship for a single channel. What ion(s) likely pass through the channel? What is its conductance? Membrane potential (mV) -100 -50 50 100 -1

6. Range of channel conductances Conductance (pS) Maximal conductance of 3 Ao diameter aqueous pore 500 Ca2+-activated ‘BK’ K+ channel 100 Nicotinic channel 20 K+ channel in axon Many channels in 5-30 pS range Na+ channel in axon 10 Aqueous pore or carrier ? Largest channels conduct 108 ions per second Fastest enzymes and transporters have turnover Rates of 105 per sec (more typically 102-104) So – ions transport must be by diffusion through aqueous pore : now confirmed by structural data. Limit of present technology 1 Store-operated Ca 2+ channels

7. Recording the activity of single channels‘Patch-clamp’ technique : Neher & Sakmann, 1976, (Nobel Prize1991) Limitation of voltage-clamp is ‘noise’ generated by large area of cell membrane. Patch-clamp overcomes this by isolating currents from tiny patch of cell membrane. Sensitive circuit then amplifies current through channel(s) in patch, while clamping voltage of pipette fixed. Current through a single channel is too small to appreciably alter Resting potential of cell, so potential across patch Remains constant.

8. A commercial patch-clamp amplifier Patching onto cultured cells under a microscope How can you see a channel to know to where to patch onto the membrane? You can’t! It’s a blind fishing expedition, and takes a lot of patience. Sometimes you might catch one channel, sometimes many channels and sometimes nothing. Getting only one channel is the ideal, as Records with more than one channel in the patch are hard to interpret. How do you know if you catch more than one channel? Sometimes you will see ‘double’ openings.

9. The ‘giga-seal’ Clearly, Rleak must be >> Rp for faithful recording. Rp is pretty much fixed (a few M Ohm) by the size of the tip (a few mm). Also, Rleak generates noise from thermal motion of ions, which decreases as Rleak increases. So, the higher Rleak can be made, the better! By using clean cell membrane (e.g. enzyme treatment to remove connective tissue, or by using cultured cells) the glass of the pipette actually sticks to the lipid membrane, forming a ‘giga-seal’ (Rleak > 1 G Ohm) Seal formation is accomplished by gently pressing the tip of the patch-pipette against the cell membrane, then applying gentle Suction.

10. Gigaseal recording configurations An unexpected, but very useful discovery was that the pipette sticks so tightly after forming a gigaseal that isolated patches of membrane can be pulled off intact from a cell. ‘whole-cell clamp’ Voltage-clamp of whole cell, but can be applied to little Cells (e.g. neurons) that are inacessible to regular voltage- clamp ‘cell-attached’ mode Study single channels in their intact cellular environment. ‘Outside-out’ excised patch Study single channels isolated from cell. Extracellular face is accessible to Bathing fluid, so can readily apply neurotransmitters or other ligands. ‘Inside-out’ excised patch. Study single channels isolated from cell. Cytosolic face is accessible to bathing fluid, so can readily apply intracellular second messengers.

11. What can patch-clamp recordings tell us? Obtain long recording with hundreds of events (openings and closings), then measure amplitudes, open and closed times for each and plot distribution histograms Distribution of single channel amplitudes Current (i) through a channel is about the same every time it opens (providing voltage is constant). However, measurement noise introduces some variability, so distributions of channel amplitudes follow a Gaussian with mean = i.

12. Mean channel open time is a characteristic of any particular type of channel, but individual openings vary randomly Long opening Short opening Random behavior gives rise to exponential distribution of open times (many short openings, few long openings) : analogous to radioactive decay Time constant of decay (t) (time to fall to 1/eof any Initial value) = mean open time Plotting on logarithmic y-axis transforms exponential distribution to linear Exponential distribution of open times on linear plot [We will talk about distribution of closed times in a future lecture]