Solid support membranes for ion channel arrays and sensors: application to rapid screening of pharmacological compounds

The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.     

Hysteresis in voltage-gated channels

Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.     

High-throughput screening for ion channel modulators

Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.     

Ionenkanäle – Einführung aus physiologischer Perspektive

Ion channels form a complex class of membrane transport proteins. They are often classified according to their selective permeability for particular ion species as well as to their gating properties, which are controlled by either membrane voltage, ligand binding or physical stimuli. Ion transport through membrane pores embedded in protein channel complexes possesses both a chemical and an electrical dimension with ion flux causing both charge separations as well as changes in ionic concentrations. This electrochemical double-nature of ion transport is reflected in the two main physiological domains of ion channel function: in excitable cells many ion channels predominately control membrane voltage to generate fast electrical signaling, while epithelial or intracellular ion channels are mainly involved in directional ion transport. Given this framework, individual channelopathies display their major deficiencies either in fast electrical signaling or ion transport itself.     

Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).     

Distribution of ion channels on taste cells and its relationship to chemosensory transduction

Summary The presence and regional localization of voltagegated ion channels on taste cells inNecturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na⁺, K⁺, or Ca²⁺ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K⁺ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na⁺ and Ca²⁺ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K⁺ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K⁺ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K⁺ conductance may underlie receptor potentials ellicted by rapid stimuli.     

Modulation and genetic identification of the M channel

Potassium channels constitute a superfamily of the most diversified ion channels, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, transmitter release, cell proliferation and cell degeneration. The M-type channel is a unique ligand-regulated and voltage-gated K(+) channel showing distinct physiological and pharmacological characteristics. This review will cover some important progress in the study of M channel modulation, particularly focusing on membrane transduction mechanisms. The K(+) channel genes corresponding to the M channel have been identified and will be reviewed in detail.It has been a long journey since the discovery of M current in 1980 to our present understanding of the mysterious mechanisms for M channel modulation; a journey which exemplifies tremendous achievements in ion channel research and exciting discoveries of elaborate modulatory systems linked to these channels. While substantial evidence has accumulated, challenging questions remain to be answered.     

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.     

Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse.

The effects of large magnitude transmembrane potential pulses on voltage-gated Na and K channel behavior in frog skeletal muscle membrane were studied using a modified double vaseline-gap voltage clamp. The effects of electroconformational damage to ionic channels were separated from damage to lipid bilayer (electroporation). A 4 ms transmembrane potential pulse of -600 mV resulted in a reduction of both Na and K channel conductivities. The supraphysiologic pulses also reduced ionic selectivity of the K channels against Na+ ions, resulting in a depolarization of the membrane resting potential. However, TTX and TEA binding effects were unaltered. The kinetics of spontaneous reversal of the electroconformational damage of channel proteins was found to be dependent on the magnitude of imposed membrane potential pulse. These results suggest that muscle and nerve dysfunction after electrical shock may be in part caused by electroconformational damage to voltage-gated ion channels.     

An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review)

Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments.     

An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review)

Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments.     

Analysis of an electrostatic mechanism for ΔpH dependent gating of the voltage-gated proton channel,HV1, supports a contribution of protons to gating charge

Voltage-gated proton channels (H_V1) resemble the voltage-sensing domain of other voltage-gated ion channels, but differ in containing the conduction pathway. Essential to the functions of H_V1 channels in many cells and species is a unique feature called ΔpH dependent gating. The pH on both sides of the membrane strictly regulates the voltage range of channel opening, generally resulting in exclusively outward proton current. Two types of mechanisms could produce ΔpH dependent gating. The “countercharge” mechanism proposes that protons destabilize salt bridges between amino acids in the protein that stabilize specific gating configurations (closed or open). An “electrostatic” mechanism proposes that protons bound to the channel alter the electrical field sensed by the protein. Obligatory proton binding within the membrane electrical field would contribute to measured gating charge. Estimations on the basis of the electrostatic model explain ΔpH dependent gating, but quantitative modeling requires calculations of the electric field inside the protein which, in turn, requires knowledge of its structure. We conclude that both mechanisms operate and contribute to ΔpH dependent gating of H_V1.     

Population patch clamp electrophysiology: a breakthrough technology for ion channel screening

Population patch clamp (PPC) is a novel high throughput planar array electrophysiology technique that allows ionic currents to be recorded from populations of cells under voltage clamp. For the drug discovery pharmacologist, PPC promises greater speed and precision than existing methods for screening compounds at voltage-gated ion channel targets. Moreover, certain constitutively active or slow-ligand gated channels that have hitherto proved challenging to screen with planar array electrophysiology (e.g. SK/IK channels) are now more accessible. In this article we review early findings using PPC and provide a perspective on its likely impact on ion channel drug discovery. To support this, we include some new data on ion channel assay duplexing and on modulator assays, approaches that have thus far not been described.     

Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles

Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.     

How voltage-gated ion channels alter the functional properties of ganglion and amacrine cell dendrites

The present study compares the structure and function of retinal ganglion and amacrine cell dendrites. Although a superficial similarity exists between amacrine and ganglion cell dendrites, a comparison between the branching pattern of the two cell types reveals differences which can only be appreciated at the microscopic level. Whereas decremental branching is found in ganglion cells, a form of non-decremental or "trunk branching" is observed in amacrine cell dendrites. Physiological differences are also observed in amacrine vs ganglion cells in which many amacrine cells generate dendritic impulses which can be readily distinguished from those of the soma, while separate dendritic impulses in ganglion cell dendrites have not been reported. Despite these differences, both amacrine and ganglion cell dendrites appear to contain voltage-gated ion channels, including TTX-sensitive sodium channels. One way to account for separate dendritic impulses in amacrine cells is to have a higher density of sodium channels and we generally find in modeling studies that a dendritic sodium channel density that is more than about 50% of that in the soma is required for excitatory, synaptic currents to give rise to local dendritic spike activity. Under these conditions, impulses can be generated in the dendrites and propagate for some distance along the dendritic tree. When the soma generates impulse activity in amacrine cells, it can activate, antidromically, the entire dendritic tree. Although ganglion cell dendrites do not appear to generate independent impulses, the presence of voltage-gated ion channels in these structures appears to be important for their function. Modeling studies demonstrate that when dendrites lack voltage-gated ion channels, impulse activity evoked by current applied to the cell body is generated at rates that are much higher than those observed physiologically. However, by placing ion channels in the dendrites at a reduced density compared to those of amacrine cells, the firing rate of ganglion cells becomes more physiological and the relationship between frequency and current (F/I relationship) can be precisely matched with physiological data. Recent studies have demonstrated the presence of T-type calcium channels in ganglion cells and our analysis suggests that they are found in higher density in the dendrites compared to the soma. This is the first voltage-gated ion channel which appears more localized to the dendrites than other cell copartments and this difference alone cries for an interpretation. The presence of a significant T-type calcium channel density in the dendrites can influence their integrative properties in several important ways. First, excitatory synaptic currents can be augmented by the activation of T-type calcium channels, although this is more likely to occur for transient rather than sustained synaptic currents because T-type currents show strong inactivation properties. In addition, T-type calcium channels may serve to limit the electrical load which dendrites impose on the spike initiation process and thus enhance the speed with which impulses can be triggered by the impulse generation site. This role whill enhance the safety factor for impulses traveling in the orthograde direction.     

Physiological and Pathological Functions of Mechanosensitive Ion Channels

Rapid sensation of mechanical stimuli is often mediated by mechanosensitve ion channels. Their opening results from conformational changes induced by mechanical forces. It leads to membrane permeation of selected ions and thereby to electrical signaling. Newly identified mechanosensitive ion channels are emerging at an astonishing rate, including some that are traditionally assigned for completely different functions. In this review, we first provide a brief overview of ion channels that are known to play a role in mechanosensation. Next, we focus on three representative ones, including the transient receptor potential channel V4 (TRPV4), Kv1.1 voltage-gated potassium (Kv) channel, and Piezo channels. Their structures, biophysical properties, expression and targeting patterns, and physiological functions are highlighted. The potential role of their mechanosensation in related diseases is further discussed. In sum, mechanosensation appears to be achieved in a variety of ways by different proteins and plays a fundamental role in the function of various organs under normal and abnormal conditions.     

Sodium channel auxiliary subunits

Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.     

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.