The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of Caenorhabditis elegans consists of a syncytium of radially orientated muscle cells that contract synchronously and rhythmically to ingest and crush bacteria and pump them into the intestine of the animal. The action potentials that support this activity are superficially similar to vertebrate cardiac action potentials in appearance with a long, calcium-dependent plateau phase. Although the pharyngeal muscle can generate action potentials in the absence of external calcium ions, action potentials are absent when sodium is removed from the extracellullar solution (Franks et al. 2002). Here we have used whole cell patch clamp recordings from the pharynx and show low voltage-activated inward currents that are present in zero external calcium and reduced in zero external sodium ions. Whilst the lack of effect of zero calcium when sodium ions are present is not surprising in view of the known permeability of voltage-gated calcium channels to sodium ions, the reduction in current in zero sodium when calcium ions are present is harder to explain in terms of a conventional voltage-gated calcium channel. Inward currents were also recorded from egl-19 (n582) which has a loss of function mutation in the pharyngeal L-type calcium channel and these were also markedly reduced in zero external sodium. Despite this apparent dependence on external sodium ions, the current was partially blocked by the divalent cations, cadmium, barium and nickel. Using single-channel recordings we identified a cation channel for which the open-time duration was increased by depolarisation. In inside-out patches, the single-channel conductance was highest in symmetrical sodium solution. Further studies are required to determine the contribution of these channels to the pharyngeal action potential.     

虎纹捕鸟蛛毒素虎纹毒素-III对美洲蜚蠊神经细胞电压门控离子通道的影响

HWTX-III是从中国虎纹捕鸟蛛Ornithoctonus huwena粗毒中分离纯化到的一种昆虫神经多肽。通过应用全细胞膜片钳技术研究了HWTX-III对美洲蜚蠊Periplaneta americana神经细胞电压门控离子通道的影响。发现HWTX-III特异性地抑制美洲蜚蠊背侧不成对中间(dorsal unpaired median, DUM）神经细胞的电压门控钠通道(IC50≈1.106 μmol/L),而对电压门控钾通道没有明显的影响。HWTX-III通过一种新型的不同于其他蜘蛛毒素的机制抑制昆虫电压门控钠通道,它不影响通道的激活与失活动力学,也不明显地漂移稳态失活曲线。HWTX-III对昆虫神经细胞电压门控钠通道的特异性与新型作用机制为研究电压门控钠通道分子结构的多样性以及开发新的安全的杀虫剂提供有用的工具。     

Threshold fluctuations in an N sodium channel model of the node of Ranvier.

Computer simulations of stochastic single-channel open-close kinetics are applied to an N sodium channel model of a node of Ranvier. Up to 32,000 voltage-gated sodium channels have been simulated with modified amphibian sodium channel kinetics. Poststimulus time histograms are obtained with 1000 monophasic pulse stimuli, and measurements are made of changes in the relative spread of threshold (RS) with changes in the model parameters. RS is found to be invariant with pulse durations from 100 microseconds to 3 ms. RS is approximately of inverse proportion to square-root of N. It decreases with increasing temperature and is dependent on passive electrical properties of the membrane as well as the single-channel conductance. The simulated RS and its independence of pulse duration is consistent with experimental results from the literature. Thus, the microscopic fluctuations of single, voltage-sensitive sodium channels in the amphibian peripheral node of Ranvier are sufficient to account for the macroscopic fluctuation if threshold to electrical stimulation.     

Ionic channels in a line of embryonal carcinoma cells induced to undergo neuronal differentiation.

The gigaseal patch clamp technique was used to investigate the electrophysiological properties of a line of embryonal carcinoma cells (PCC4) that were induced to undergo neuronal differentiation. A large increase in number of voltage-dependent potassium and sodium channels was observed during differentiation. The pharmacology and kinetics of the macroscopic sodium and potassium currents in the differentiated cells closely resembled those of the rapid inward sodium current and the delayed rectifier, respectively. The kinetic behavior of single-channel potassium currents was consistent with the properties of the macroscopic delayed rectifier current.     

Application of the ensemble nonequilibrium response spectroscopy to shaker potassium channel gating

Standard electrophysiology techniques study relaxation transients in voltage-gated ion channels generated by discrete voltage steps. The nonequilibrium response spectroscopy involves analyzing responses to fluctuating potentials. We apply the ensemble NRS method to gating kinetics of Shaker potassium ion channels. We evaluate various proposed Markov models of channel gating from the nonequilibrium response viewpoint. These new NRS protocols can be used to test otherwise indistinguishable models or improve estimates for parameters of channel kinetics models.     

Single-channel recording of ligand-gated ion channels

Electrophysiological recording of single-channel currents is the most direct method available for obtaining detailed and precise information about the kinetic behavior of ion channels. A wide variety of cell types can be used for single-channel recording, but to obtain the highest resolution of the briefest channel opening and closing events, low-noise recordings, coupled with a minimal filtering frequency, are required. Here, we present a protocol designed to help those with some electrophysiological expertise who wish to explore the properties of native and recombinant single ligand-gated ion channels. We have focused on the practical aspects of recording single GABA channels from cell-attached and outside-out patches and also introduced some of the preliminary considerations that are necessary for the analysis of single-channel data, including an introduction to single-channel analysis software.     

Optimal estimation of ion-channel kinetics from macroscopic currents

Markov modeling provides an effective approach for modeling ion channel kinetics. There are several search algorithms for global fitting of macroscopic or single-channel currents across different experimental conditions. Here we present a particle swarm optimization(PSO)-based approach which, when used in combination with golden section search (GSS), can fit macroscopic voltage responses with a high degree of accuracy (errors within 1%) and reasonable amount of calculation time (less than 10 hours for 20 free parameters) on a desktop computer. We also describe a method for initial value estimation of the model parameters, which appears to favor identification of global optimum and can further reduce the computational cost. The PSO-GSS algorithm is applicable for kinetic models of arbitrary topology and size and compatible with common stimulation protocols, which provides a convenient approach for establishing kinetic models at the macroscopic level.     

Determining the target of membrane sterols on voltage-gated potassium channels

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel K_v1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing K_v1.3 and K_v10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.     

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels?

Ion channels are protein molecules, which can assume distinct open and closed conformational states, a phenomenon termed ion channel kinetics. The transitions from one state to another depend on the potential energy barrier that separates those two states. Therefore, it is rational to suppose that electromagnetic waves could interact with this barrier and induce changes in the rate transitions of this kinetic process. Our aim is to answer the question: can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels? We simulated the effects of the low and high frequency electromagnetic waves on the sodium and potassium channels of the giant axon of Loligo. The key parameter measured was the fractional open time (fv), because it reflects the voltage dependence of the kinetics of channels. The electromagnetic radiations induced the following changes in the kinetics of the potassium and sodium channels: i/ low frequency waves kept the potassium channel 50% of the time open independent on the mean voltage applied through the membrane; ii/ a gradual inhibition of the inactivation on the sodium channel, when the amplitudes of the low frequency waves were increased; iii/ high frequency waves on the potassium channel, decreased both Vo (voltage in which the channel stays 50% open) and the steepness of fv (d fv/dV) as the amplitudes of the waves increased, and iv/ high frequency and low amplitude radiations on the sodium channel decreased the maximum value of fv (in relation to control), while high amplitudes increased this value. In conclusion, high and low frequency electromagnetic radiations were able to change the kinetics of the potassium and sodium channels in a squid giant axon model.     

海南捕鸟蛛毒素-Ⅵ，一种新型的抑制昆虫电压门控钠通道失活的狼蛛神经毒素

通过阳离子交换和反相HPLC柱层析从海南捕鸟蛛（Ornithoconus hainana）粗毒中分离到一种新型的神经毒素,海南捕鸟蛛毒素-Ⅵ(HNTX-Ⅵ), 由34个氨基酸残基组成,含有6个保守的半胱氨酸残基. 运用全细胞膜片钳技术,研究了HNTX-Ⅵ对电压门控钠通道的影响.先前从海南捕鸟蛛粗毒中分离到的几种毒素,具有抑制哺乳动物钠通道激活的特性.本文研究结果表明,HNTX-Ⅵ能以类似于δ-atractoxins作用方式延缓蜚蠊背侧不成对中间(dorsal unpaired median,DUM）神经细胞的钠通道的失活,且导致钠通道稳态失活变得不完全,在预钳制电压大于-55 mV时形成不完全失活结构. HNTX-Ⅵ的这种新的功能不仅为探索钠通道的门控机制提供了有用的工具,也为开发新的安全的杀虫剂提供理论基础.     

A historical biophysical dogma vs. an understanding of the structure and function of voltage-gated tetrameric ion channels. A review

The outstanding work of several eminent biophysicists has allowed the functional features of voltage-gated tetrameric ion channels to be disclosed using ingenious and sophisticated electrophysiological techniques. However, the kinetics and mechanism underlying these functions have been heavily conditioned by an arbitrary interpretation of the groundbreaking results obtained by Hodgkin and Huxley (HH) in their investigation of sodium and potassium currents using the voltage clamp technique. Thus, the heavy parametrization of their results was considered to indicate that any proposed sequence of closed states terminates with a single open state. This ‘dogma’ of HH parametrization has influenced the formulation of countless mechanistic models, mainly stochastic, requiring a high number of free parameters and of often unspecified conformational states. This note aims to point out the advantages of a deterministic kinetic model that simulates the main features of tetrameric ion channels using only two free parameters by assuming their stepwise opening accompanied by a progressively increasing cation flow. This model exploits the electrostatic attractive interactions stemming from the charge distribution shared by all tetrameric ion channels, providing a close connection between their structure and function. Quite significantly, a stepwise opening of all ligand-gated tetrameric ion channels, such as glutamate receptors (GluRs), with concomitant ion flow, is nowadays generally accepted, not having been influenced by this dogma. This provides a unified picture of both voltage-gated and ligand-gated tetrameric ion channels.     

Estimation of ion channel kinetics from fluctuations of macroscopic currents

For single channel recordings, the maximum likelihood estimation (MLE) of kinetic rates and conductance is well established. A direct extrapolation of this method to macroscopic currents is computationally prohibitive: it scales as a power of the number of channels. An approximated MLE that ignored the local time correlation of the data has been shown to provide estimates of the kinetic parameters. In this article, an improved approximated MLE that takes into account the local time correlation is proposed. This method estimates the channel kinetics using both the time course and the random fluctuations of the macroscopic current generated by a homogeneous population of ion channels under white noise. It allows arbitrary kinetic models and stimulation protocols. The application of the proposed algorithm to simulated data from a simple three-state model on nonstationary conditions showed reliable estimates of all the kinetic constants, the conductance and the number of channels, and reliable values for the standard error of those estimates. Compared to the previous approximated MLE, it reduces by a factor of 10 the amount of data needed to secure a given accuracy and it can even determine the kinetic rates in macroscopic stationary conditions.     

Invariant manifold reductions for Markovian ion channel dynamics

We show that many Markov models of ion channel kinetics have globally attracting stable invariant manifolds, even when the Markov process is time dependent. The primary implication of this is that, since the dimension of the invariant manifold is often substantially smaller than the full master equation system, simulations of ion channel kinetics can be substantially simplified, with no approximation. We show that this applies to certain models of potassium channels, sodium channels, ryanodine receptors and IP₃ receptors. We also use this to show that the original Hodgkin–Huxley formulations of potassium channel conductance and sodium channel conductance are the exact solutions of full Markov models for these channels.      

Voltage-gated cation and anion channels in mammalian Schwann cells and astrocytes

This article reviews some recent studies on the voltage-gated ion channels in the plasmalemma of the satellite cells of the peripheral and central nervous systems. Following the finding that rabbit Schwann cells exhibit a large binding capacity for saxitoxin (Ritchie and Rang, 1983) electrophysiological studies have shown that these cells not only express plasmalemmal voltage-gated sodium channels but also voltage-gated potassium channels (Chiu, Shrager and Ritchie, 1984; Shrager, Chiu and Ritchie, 1985). Whole-cell recording with the patch-clamp method reveals that the properties of these two kinds of channel are quite similar to those of the corresponding channels in the nodal axolemma, except that the peak current-voltage relation of the sodium channels is shifted about 30 mV in the depolarizing direction. Similar voltage-gated sodium and potassium channels exist in rat cultured astrocytes (Bevan et al., 1985). Furthermore, the outward current on depolarization in astrocytes has a component other than that carried in the potassium channels. About 75% of the total outward current is blocked by external TEA or internal caesium; and it is presumed to be a potassium current. The remainder, however, is insensitive to these potassium channel blocking agents; but it is abolished by exposure to the two stilbene sulphonates, DIDS and SITS (Gray and Ritchie, 1986). This remaining current persists in the presence of the large organic cation N-methyl-(+)-glucamine in the patch pipette. It is, however, reduced when the chloride of the external medium is replaced by methyl-sulphate, sulphate, or isethionate; and it is abolished when the external anion is gluconate (M.W., 190). The TEA-insensitive component of outward current in astrocytes thus seem to involve an influx of chloride ions through a voltage-gated channel whose diameter is such that anions larger than gluconate cannot pass. The current in the channels is neglible at potentials more negative than about -40 mV.     

Phylogeny of ion channels: clues to structure and function

Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types in modern-day animals. However, they represent the result of many millions of years of evolution of a family of ion channel proteins that are also found in prokaryotes and diverse eukaryotes, and probably exist in all life forms. This review traces the evolution of ion channels, with particular emphasis on the factors and evolutionary pathways that may have given rise to voltage-gated potassium (K+), calcium (Ca2+), and sodium (Na+) channels. The review also highlights the utility of comparing phylogenetically distinct versions of the same protein as a means to better understand the structure and function of proteins.     

Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes

Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.