Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

电压门控Na^＋通道亚型及其功能——新的研究热点

依靠现代分子生物学技术及电生理的记录,探讨各种Ｎａ＾＋通道亚型在中枢与周边神经系统以及一些非兴奋性组织细胞中的分布,表达,突变及其对信息调控的功能特征,已成为当今神经生物学等学科发展中的一个研究新热点,本文将侧重对有关哺乳动物Ｎａ＾＋通道亚型的分类,在不同组织细胞中的分布及其表达调控的功能机制等一些研究进展做一简要的回眸。     

电压门控钠通道的变异与机体疾患

通道病理学是当今国际学术发展中一门新兴学科。本文将针对有关电压门控钠通道的变异所导致的机体疾患,如高血钾性周期性麻痹,先天性肌强直等骨骼肌疾患,ＬＱＴ３,原发笥心室纤颤等心脏病及其所涉及的钠通道突变体,通道的突变位点和电生理性质等一些研究资料与进展作一概括介绍。     

Current–voltage–time records of ion translocating enzymes

Membrane currents, as non-linear functions of membrane voltage, V, and time, t, can be recorded quickly by triangular V protocols. From the differences, dI(V,t), of these relationships upon addition of a putative substrate of a charge-translocating membrane protein, the I(V,t) relationships of the transporter itself can be determined. These relationships likely comprise a steady-state component, I_a(V), of the active transporter, and a dynamic component, p_a(V,t), of its V- and time-dependent activity, p_a. Here, the steady-state component is modeled by a central reaction cycle, which senses a fraction _tr of the total V, whereas 1–_tr can be assigned to an inner and outer pore section with _i and _o, respectively (_i+_tr+_o = 1). For the enzymatic cycle, fast binding/debinding is assumed, plus V-sensitive and -insensitive reaction steps which may become rate limiting for charge translocation. At given substrate concentrations, I_a(V) is defined by eight independent system parameters, including a coefficient for the barrier shape of charge translocation. In ordinary cases, the behavior of p_a(V,t) can be described by two rate constants (for activation and inactivation) and their respective V-sensitivity coefficients. Here, the effects of the individual system parameters on I(V,t) from triangular V-clamp experiments are investigated systematically. The results are illustrated by panels of typical curve shapes for non-gated and gated transporters to enable a first classification of mechanisms. We demonstrate that all system parameters can be determined fairly well by fitting the model to experimental data of known origin. Applicability of the model to channels, pumps and cotransporters is discussed.     

Interpretation and Optimization of Absorbance and Fluorescence Signals from Voltage-sensitive Dyes

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density (OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.     

Potassium channels

The atomic structures of K+ channels have added a new dimension to our understanding of K+ channel function. I will briefly review how structures have influenced our views on ion conduction, gating of the pore, and voltage sensing.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

Three Types of Membrane Excitations in the Marine Diatom Coscinodiscus wailesii

Three types of electrical excitation have been investigated in the marine diatom Coscinodiscus wailesii. I: Depolarization-triggered, transient Cl⁻ conductance, G _Cl (t), followed by a transient, voltage-gated K⁺ conductance, G _K , with an active state a and two inactive states i ₁ and i ₂ in series (a-i ₁-i ₂). II: Similar G _Cl (t) as in Type-I but triggered by hyperpolarization; a subsequent increase of G _K in this type is indicated but not analyzed in detail. III: Hyperpolarization-induced transient of a voltage-gated activity of an electrogenic pump (i ₂-a-i ₂), followed by G _Cl (t) as in Type-II excitations. Type-III with pump gating is novel as such. G _Cl (t) in all types seems to reflect the mechanism of InsP⁻ ₃ and Ca²⁺-mediated G _Cl (t) in the action potential in Chara (Biskup et al., 1999). The nonlinear current-voltage-time relationships of Type-I and Type-III excitations have been recorded under voltage-clamp using single saw-tooth command voltages (voltage range: −200 to +50 mV, typical slope: ±1 Vs⁻¹). Fits of the corresponding models to the experimental data provided numerical values of the model parameters. The statistical significance of these solutions is investigated. We suggest that the original function of electrical excitability of biological membranes is related to osmoregulation which has persisted through evolution in plants, whereas the familiar and osmotically neutral action potentials in animals have evolved later towards the novel function of rapid transmission of information over long distances. Received: 2 December 1999/Revised: 3 March 2000     

Activation and Inactivation of Mechanosensitive Currents in the Chick Heart

The behavior of MS channels in embryonic chick ventricular myocytes activated by direct mechanical stimulation is strongly affected by inactivation. The amplitude of the current is dependent not only on the amplitude of the stimulus, but also the history of stimulation. The MS current inactivation appears to be composed of at least two contributions: (i) rearrangement of the cortical tension transducing elements and (ii) blocking action of an autocrine agent released from the cell. With discrete mechanical stimuli, the MS current amplitude in the second press of a double press protocol was always smaller than the amplitude of the first MS current. Occasionally, a large MS current occurred when the cell was first stimulated, but subsequently the cell became unresponsive. For a series of stimuli of varying amplitudes, the order in which they were applied to the cell affected the size of the observed MS current for a given stimulus magnitude. When continuous sinusoidal stimulation was applied to the cells, the MS current envelope either reached a steady state, or inactivated. With sinusoidal stimulation, the MS response could be enhanced or restored by simple perfusion of fluid across the cell. This suggests that mechanical stimulation of the cells produces an autocrine inhibitor of MS channels as well as resulting in cortical rearrangement. Received: 7 July 1999/Revised: 26 October 1999     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000