Harold Lecar

Electrically gated ionic channels in lipid bilayers

The generation of action potentials in nerve and muscle requires cell membranes with steeply voltage-dependent ionic permeabilities. The voltage-dependent, ion-selective pathways responsible for excitation have been characterized for numerous excitable tissues such as nerve axon, muscle, electric organ, algae and epithelia (Aidley, 1971; Hodgkin, 1964; Cole, 1968). The process of activating ionic pathways by some stimulus, such as a change in membrane potential, is called gating.

Theory of Threshold Fluctuations in Nerves: I. Relationships between Electrical Noise and Fluctuations in Axon Firing

Relations describing threshold fluctuation phenomena in nerves are derived by calculating the approximate response of the Hodgkin-Huxley (HH) axon to electrical noise. We use FitzHughs reduced phase space approximation and describe the dynamics of a noisy nerve by a two-dimensional brownian motion. The theory predicts the functional form and parametric dependence of the relation between probability of firing and stimulus strength. Expressions are also obtained for the firing probability as a function of stimulus duration and for the distribution of latency times as a function of stimulus strength.

Noise analysis of the glutamate-activated current in photoreceptors

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.

Theory of Threshold Fluctuations in Nerves: II. Analysis of Various Sources of Membrane Noise

Threshold fluctuations in axon firing can arise as a result of electrical noise in the excitable membrane. A general theoretical expression for the fluctuations is applied to the analysis of three sources of membrane noise: Johnson noise, excess 1/f noise, and sodium conductance fluctuations. Analytical expressions for the width of the firing probability curve are derived for each of these noise sources. Specific calculations are performed for the node of Ranvier of the frog, and attention is given to the manner in which threshold fluctuations are affected by variations of temperature, ion concentrations, and the application of various drugs. Comparison with existing data suggests that threshold fluctuations can best be explained by sodium conductance fluctuations. Additional experiments directed at distinguishing among the various noise sources are proposed.

Electrostatic Model of S4 Motion in Voltage-Gated Ion Channels

The S4 transmembrane domain of the family of voltage-gated ion channels is generally thought to be the voltage sensor, whose translocation by an applied electric field produces the gating current. Experiments on hSkMI Na(+) channels and both Shaker and EAG K(+) channels indicate which S4 residues cross the membrane-solution interface during activation gating. Using this structural information, we derive the steady-state properties of gating-charge transfer for wild-type and mutant Shaker K(+) channels. Assuming that the energetics of gating is dominated by electrostatic forces between S4 charges and countercharges on neighboring transmembrane domains, we calculate the total energy as a function of transmembrane displacement and twist of the S4 domain. The resulting electrostatic energy surface exhibits a series of deep energy minima, corresponding to the transition states of the gating process. The steady-state gating-charge distribution is then given by a Boltzmann distribution among the transition states. The resulting gating-charge distributions are compared to experimental results on wild-type and charge-neutralized mutants of the Shaker K(+) channel.

Noise analysis of ion channels in non-space-clamped cables: Estimates of channel parameters in olfactory cilia

Ion channels in the cilia of olfactory neurons are part of the transduction machinery of olfaction. Odorant stimuli have been shown to induce a biphasic current response, consisting of a cAMP-activated current and a Ca(2+)-activated Cl- current. We have developed a noise analysis method to study ion channels in leaky cables, such as the olfactory cilium, under non-space-clamp conditions. We performed steady-state noise analysis on ligand-induced currents in excised cilia, voltage-clamped at input and internally perfused with cAMP or Ca2+. The cAMP-activated channels analyzed by this method gave results similar to those of single-channel recordings (gamma = 8.3 pS). Single-channel currents have not yet been recorded for the Ca(2+)-activated Cl- channels. Using our noise analysis method, we estimate a unit conductance, gamma = 0.8 pS, for these channels. The density of channels was found to be approximately 70 channels/micron2 for both channel species.

SQUID AXON MEMBRANE RESPONSE TO WHITE NOISE STIMULATION

The current from a white noise generator was applied as a stimulus to a space-clamped squid axon in double sucrose gap. The membrane current and the voltage response of the membrane were then amplified, recorded on magnetic tape, and the stimulus was cross-correlated with the response. With subthreshold stimuli, a cross-correlation function resembling that obtained from a resonant parallel circuit is obtained. As the intensity of the input noise is increased, the cross-correlation function resembles that obtained from a less damped oscillatory circuit. When the noise intensity is further increased so that an appreciable frequency of action potentials is observed, an additional component appears in the experimental cross-correlogram. The subthreshold cross-correlogram is analyzed theoretically in terms of the linearized Hodgkin-Huxley equations. The subthreshold axon approximates a parallel resonant circuit. The circuit parameters are temperature dependent, with resonant frequency varying from approximately 100 Hz at 10 degrees C to approximately 250 Hz at 20 degrees C. The Q(10) of the resonant frequency is equal to 1.9. These values are in agreement with values found previously for subthreshold oscillations following a single action potential.

Ionic channels in the plasma membrane of Schizosaccharomyces pombe: Evidence from patch-clamp measurements

Patch-clamp studies of the yeastSchizosaccharomyces pombe reveal that the plasma membrane contains a voltage-gated channel mildly selective for potassium over sodium, lithium, and chloride. The channel exhibits several conductances with a maximum of 153 pS. The channel gates in the region of physiologically relevant voltages, being closed at hyperpolarizing and open at depolarizing voltages. It is not inhibited by tetraethylammonium, quinine, or quinidine applied from the cytoplasmic side of the membrane; similarly, ATP and stretch have no effect. The frequency of its occurrence in patches implies that about 35 channels of this kind are present in the plasma membrane of a single cell.

Detection of Molecular Motion in Lyophilized Myelin by Nuclear Magnetic Resonance

Proton resonance spectroscopy was used to determine the state of the hydrocarbon regions in lyophilized and resuspended samples of nerve myelin. Measurements of the resonance line width indicate considerable freedom of motion within the hydrocarbon moiety of the myelin samples. Sharp thermal transitions of the line width were observed, suggesting that lyophilized myelin is in a liquid crystalline state.

Patch-Clamp Techniques and Analysis

Publisher Summary Patch-clamp technique revolutionized electrophysiology by revealing the activity of individual molecular ion channels involved in electrical signaling in excitable cells. This chapter focuses on the technique and its application in studies of single channels or unitary conductance. It describes different variations of the patch-clamp technique, that is, cell-attached, inside-out, and outside-out, and its modifications as used in the study of single-channel currents. It also explains the more commonly used application of whole-cell voltage clamp and its variation, and the perforated-patch methods that examine macroscopic currents. Although whole-cell recording provides information of the gating properties and kinetic characteristics of the channel protein, single-channel recordings allow a more detailed evaluation of the kinetic properties of the channel protein. The different variations or modes of patch recordings permit the experimenter to alter the environment and examine the gating and behavior of a single-channel protein. Another variation of the patch-clamp technique, the whole-cell tight-seal voltage clamp, proves to be particularly useful for the study of macroscopic currents. As this method is applicable to small cells, it opens up a new vista for experimentation, allowing experimenters to examine currents in cells that were previously not accessible to recording with conventional sharp-tipped electrodes.