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2005-06 Electrophysiology Membrane potential (Kandel Ch 7 – pp 125) Information within and between neurons: by electrical and chemical signals. Receptor, synaptic and action potentials across the membrane: a. Transient electrical signals that carry time- and intensity-sensitive information (how qualitysensitive information is transferred?). b. Produced by changes in the current flow into and out of the cell. c. Current is controlled by ion channels in the membrane. Ion channels: a. Resting – normally open and maintain the resting membrane potential. b. Gated – closed during resting potential and opening regulated by changes in: * Membrane potential - voltage dependant, * Ligand binding - associated with receptor, * Membrane stretch – sensory receptor. Resting membrane potential: a. Due to separation of charges across the membrane. b. Vm = Vin – Vout = ~ -60 mV c. Not a stable condition since frequent opening of ion channels. Electrical current flows into and out of the cell: a. Carried by ions (positive cations and negative anions). Normal current in socket is carried by electrons. b. Direction of current is defined as direction of net movement of positive ions. c. Current in = depolarization; Current out = hyperpolarization. Changes in membrane potential: a. Electrotonic (graded) – passive changes in membrane potential if not leading to opening of gated channels. b. Electrotonic – hyperpolarizations and small depolarizations. c. Action potentials – opening of voltage sensitive channels at certain voltage threshold. Resting membrane potential: a. Asymmetric distribution of ion across the membrane b. Out – more Na+ and Clc. In – more K+ and organic anions ANernst equation: a. Used to find the equilibrium potential of any ion present on both sides of the membrane permeable to that ion. b. K+ = -75 mV c. Na+ = +55 mV d. Cl- = -60 mV e. A- = membrane not permeable Ion Equilibrium Potential = (gas constant * temp) / (ion valence * Faraday constant) * ln (ion concentration out / ion concentration in) = Ion constant * ln (ion concentration out / ion concentration in) Equilibrium potential: a. The electrical force related to potential gradient equals the chemical force related to concentration gradient. b. Ion flux = (electrical force + chemical force) * membrane conductance. Resting channels – resting potential: a. Electrophysiology and flux studies using radioactive tracers. b. Nerve cells at rest are permeable to Na (low amount of resting channels), Cl, and K (high amount). Implication for resting potential ??? c. Membrane is not permeable to A anions. d. Fig. 7-4 (pp 130). e. Resting potential affected by K equilibrium potential because of the many resting channels (-75mV). f. Resting potential is slightly depolarized from the K equilibrium potential due to influx of Na. But only slightly because not too many resting Na channels. g. Influx of Na to the cell reduces the electrical inward force on K and therefore K starts to flow out, thus compensating for the Na influx, but at new resting potential (-60 instead of -75mV). Wilson & Kawaguchi, 1996 (J. Neurosci., 16: 2397-2410) Active pumping of the ions: a. The outflow of K and the inflow of Na is balanced at the new resting potential. b. This steady leak of ions across the membrane has to be compensated to prevent decrease of the concentration gradients. c. This is achieved by the Na-K active pump using ATP derived energy. d. Thus, at the resting potential the cell is not at equilibrium (there is ion flow) but rather in a steady state: continuous ion flow counterbalanced by Na-K pump (Na/K = 3/2, hyperpolarization effect). e. The pump has binding sites for Na and ATP on the intra-cellular surface of the membrane and for K on the extra-cellular surface. f. The resting potential is just about the equilibrium potential of the Cl and therefore it has no effect on the resting potential. Action potential: a. Once the membrane potential exceeds some threshold value, voltage gated Na channels open rapidly. b. The result is depolarization toward the Na equilibrium potential +55mV. c. The potential does not reach +55 mV since there is constant efflux of K and influx of Cl. d. This mechanism of spike is terminated by gradual inactivation of the voltage gated Na channels and by opening of voltage gated K channels. This creates a net efflux of positive charges from the cell until the cell reaches back the resting potential. The action potential (Kandel Ch 9 – pp 150) Action potential a. Action potential is generated and propagated by voltage-gated ion channels. It is an active signal conductance over long distance without attenuation of amplitude (unlike the passive current). b. Ion conductance across the membrane increases during the action potential. c. Na influx is responsible for the rising phase of the action potential due to a transient increase in membrane permeability to Na. What is the effect of increase Na concentration? d. K efflux is responsible for the falling phase of the action potential due to a transient increase in membrane permeability to K. What is the effect of the increase in K concentration? How the properties of the voltage-gated Na and K channels are studied? The intention was to vary the voltage across the membrane and observe the changes in Na and K conductance. a. Before the voltage clamp technique was developed, there was a problem to stabilize the membrane potential because of a positive feedback: experimental depolarization opens the Na voltagegated channels which lead to inward flux of Na which cause depolarization and so on, until the membrane reaches the peak of the action potential. b. The voltage clamp technique prevents the opening and closing of the voltage-gated ion channels upon change of the membrane potential. It does so by injecting into the cell a current that is equal and opposite to the current flowing through the voltagegated channels. c. The current that has to be injected to keep the membrane potential stable is a direct measure of the ionic current through the ionic channels. Voltage clamp technique for studying membrane currents of a squid axon. The device permits the simultaneous measurement of the current needed to keep the cell at a given voltage (4). Therefore, the voltage clamp technique can indicate how membrane potential influences ionic current flow across the membrane. This information gave Hodgkin and Huxley the key insights that led to their model for action potential generation. Experiment with voltage clamp technique (Fig 9-3, p:153; Box 9-1, p.152) a. Experiment starts at resting potential. b. 10mV depolarization pulse through the voltage clamp electrode leads to transient outward capacitance current followed by a leakage current. Capacitance current (related to the lipid layer) is starting only with a change in the membrane potential. It is instantaneous as it discharges/charges through the resistance of the membrane. It is much faster than the dynamics of the ionic currents. Leakage current is mainly through the resting ion channels, mainly the K channels (highest permeability), but also Na and Cl resting channels. c. 20 mV depolarization pulse leads to larger currents. First the outward capacitance discharge current followed by outward leakage current. Then inward and then outward current, most likely due to conductance through Na and K voltage gated channels. d. To separate the time course of the last two currents, the axon is treated with selective blockers of the Na or K channels. e. Such experiments demonstrated simultaneous fast opening of the Na channels and slower opening of the K channels. f. Such experiments are then repeated at different voltage depolarization steps. Refinement of the voltage clamp technique is the patch clamp technique for recording in single ion channels (Box 6-1, p.111). http://www.science-display.com/patchclamp.html The pipette electrode is filled with a ligand (for example) that activates the channel to record a transmitter-gate channels in the membrane. Four configurations in patch clamp measurements of ionic currents. Back to spike mechanisms: Fig 9-6 and Fig 9-7 (p 156) shows that depolarization pulses cause fast increases in conductance of Na voltage dependent channels (inward current) which are inactivated even if the depolarizing pulses are still on. The same depolarizing pulses are slowly increasing the conductance of voltage dependent K channels which stay open as long as the pulses are on. The above sequence of Na and K channels opening define the shape and the duration of the action potential. A small riddle is why the action potential is all or none phenomena inspite of the fact that the voltage dependent Na channels are opened already with subthreshold depolarizations. The answer is that until certain depolarization level the inward Na is opposed by outward K and Cl currents. However at certain level of depolarization the Na current is stronger than the other currents because of the great sensitivity of Na channels to voltage and its fast opening dynamics. At this point the net current of Na is stronger than the other currents and produces a regenerative depolarization. Life after the spike: Fig 9-9 shows the complex votage dependent dynamics of the Na channel. It has two gates: activation gate and inactivation gate. Only at the depolarization level the two gates are open and therefore produce the rising phase of the spike. As the inactivation Na gate is very slow to open, it causes an absolute refractory period after the spike termination. During this period no depolarization current can produce a new spike. Residual opening of the voltage dependent K channels after the spike cause the relative refractory period during which stronger depolarization is needed to produce a spike (see Fig 9-6 for the residual opening of the K channels after the depolarizing current is already off). The story of the Ca2+: Unlike other ions, the concentration of Ca which is initially very low in the cell, is increasing significantly during depolarization due to the opening of voltage-gated Ca channels. The effects of the inward flow of Ca are limited since Ca increases the opening of the K channels (efflux of K) and closing of the Ca channels by the inside Ca concentration. Eventually the influx of Ca serves as a second messenger for changing the properties of some channels. Excitability at different regions of the neuron: In some neurons the dendrites have voltage-gated ion channels, such as Ca, K and also Na. activation of these channels modify the passive, electrotonic conduction of synaptic potentials. In some of these neurons the spike can be propagated from the axon hillock (the integration zone due to highest concentration of voltage gated Na channels) back into the dendrites. This kind of mechanism was shown recently to be involved in learning phenomena and explains how certain region of the dendrite may be potentiated. Excitability properties vary among neurons How the neuron responds to synaptic input is determined by the proportions of different types of voltage-gated channels in the neuron's integrative and trigger (integrate and fire) zone. Some cells respond with a single spike others with burst of spikes. Various response options are shown in Fig 9-11. Particularly interesting is section B and C. Section B shows the generation of delayed burst activity after prolonged inhibition which causes the voltage gated Ca channel to recover from inhibition. Section C shows repeated bursting (contribution to normal and epileptic EEG rhythmic patterns). This pattern is also caused by strong effects of Ca channel activated during Na influx and closed between the bursts, thus allowing hyperpolarization. END