Chiral aspects of drug action at ion channels: a commentary on the stereoselectivity of drug actions at voltage-gated ion channels with particular reference to verapamil actions at the Ca2+ channel.

Ion channels may be considered as pharmacological receptors possessing specific drug binding sites with defined structure-activity relationships. Accordingly drug binding to ion channels is stereoselective. Interpretation of this stereoselectivity may be complex because of the existence of differences in affinity and access to different channel states. Such state-dependent interactions may give rise to quantitative and qualitative differences in stereoselectivity. The implications of such differences are reviewed for drug action at Na+, K+ and Ca2+ channels. Detailed attention is paid to the actions of verapamil enantiomers in the cardiovascular system where activities differ in vascular and cardiac tissues because of state-dependent interactions and stereoselective first-oass metabolism.     

Ion channels in cancer: future perspectives and clinical potential

Ion transport across the cell membrane mediated by channels and carriers participate in the regulation of tumour cell survival, death and motility. Moreover, the altered regulation of channels and carriers is part of neoplastic transformation. Experimental modification of channel and transporter activity impacts tumour cell survival, proliferation, malignant progression, invasive behaviour or therapy resistance of tumour cells. A wide variety of distinct Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels have been implicated in tumour growth and metastasis. Further experimental information is, however, needed to define the specific role of individual channel isoforms critically important for malignancy. Compelling experimental evidence supports the assumption that the pharmacological inhibition of ion channels or their regulators may be attractive targets to counteract tumour growth, prevent metastasis and overcome therapy resistance of tumour cells. This short review discusses the role of Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels in tumour growth and metastasis and the therapeutic potential of respective inhibitors.     

Defining the roles of Ca2+ — permeable channels in sperm

Ion channels exert a vital role in the dialogue between male and female gametes and thus in the generation of new individuals in many species. Intracellular Ca²⁺ is possibly the key messenger between gametes. Different Ca²⁺-permeable channels have been detected in the plasma membrane and in the organelle-like acrosome membrane of sperm, which play vital roles in determining sperm fertilizing ability. Recent reports from several laboratories have adequately documented that the Ca²⁺-permeable channels of a sperm control a variety of functions ranging from motility to the acrosome reaction. In this article, we have reviewed the data from our and other laboratories, and have documented the mechanisms of different Ca²⁺-permeable channels involved in the fertilization event.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

Membrane-delimited cell signaling complexes: Direct ion channel regulation by G Proteins

Summary Ion channels are signaling molecules and by them-selves perform no work. In this regard they are un like the usual membrane enzyme effectors for G proteins. The pathways of G protein receptor, G protein and ion channels are, therefore, purely infor mational in function. Because a single G protein may have several ion channels as effectors, the effects should be coordinated and this seems to be the case. Inhibition of Ca²⁺ current and stimulation of K⁺ currents would have a greater impact than either alone. Additional flexibility is provided by spontane ous noise in the complexes of G protein receptor, G protein, and ion channel. By having a non-zero setpoint, the range of control is extended and the responses become bi-directional.     

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na⁺ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na⁺ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF⁻¹. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na⁺ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a “window-current” occurred, where 10–18% of the Na⁺ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na⁺ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain. Accepted: 1 October 1998     

Imbalance and dysfunction of transient receptor potential channels contribute to the pathogenesis of hypertension

Intracellular Ca²⁺ homeostasis is essential for vascular function and blood pressure regulation. Because of their unique roles in regulating intracellular Ca²⁺ concentration and vascular function, a novel class of non-selective cation channels, called transient receptor potential (TRP) channels, have emerged at the frontier of hypertension research. Based on their role in vasculature function regulation, TRP channels can be divided into two functional subtypes: one that participates in vasoconstriction and one that participates in vasodilatation. A functional imbalance of these two subtypes of TRP channels may disturb intracellular calcium ([Ca²⁺]i) homeostasis, and the consequent vascular dysfunction may contribute to the development of hypertension. The potential of these TRP channels as novel pharmacological targets for the treatment of human hypertension is of great interest.     

Two types of modified cardiac Na+ channels after cytosolic interventions at the α-subunit capable of removing Na+ inactivation

Failure of inactivation is the typical response of voltage-gated Na⁺ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the α-subunit. The present patch clamp experiments with cardiac Na⁺ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na⁺ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481–1496 of the cardiac Na⁺ channel α-subunit) eliminated fast Na⁺ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at –40 mV). NBA-modified and iodate-modified Na⁺ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate P_o of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na⁺ channels: after cytosolic proteolysis with α-chymotrypsin, trypsin or pronase, mean P_o during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100–200 ms at –40 mV, as a minimum) or was virtually non-operating. In-vitro cleavage of the synthetic linker sequence 1481–1496 confirmed that this part of the α-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na⁺ channels are based on a structural alteration of still another region which is also involved in Na⁺ inactivation, besides the linker between domains III and IV of the α-subunit. Endogenous peptidases such as calpain did not affect Na⁺ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na⁺ channels, mode-switching to a non-inactivating mode. Received: 25 May 1996 / Accepted: 12 September 1996     

Plant K+ Channels: Similarity and Diversity

In this review we compared the electrophysiological properties of plant K⁺ uptake channels from different plants and tissues. Taking into account the detailed knowledge of K⁺ channel properties, which has emerged since the application of the patch-clamp technique on plant cells, as well as results from our recent studies we were able to extract features common among plant K⁺ channels. In addition, we focused on the diversity that could create plant or tissue-specificity. Functional fingerprints for the voltage-dependent K⁺ uptake channels were generated on the basis of their voltage-dependence, kinetics, permeability, conductance and pharmacology as well as regulation of K⁺ channels studied in their natural environment and cloned channels in heterologous expression systems. Finally, sequence information on plant and animal K⁺ channels cloned so far was used to identify structural motifs that may be related to functional phenotypes.     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

The hidden potential of lysosomal ion channels: A new era of oncogenes

Lysosomes serve as the control centre for cellular clearance. These membrane-bound organelles receive biomolecules destined for degradation from intracellular and extracellular pathways; thus, facilitating the production of energy and shaping the fate of the cell. At the base of their functionality are the lysosomal ion channels which mediate the function of the lysosome through the modulation of ion influx and efflux. Ion channels form pores in the membrane of lysosomes and allow the passage of ions, a seemingly simple task which harbours the potential of overthrowing the cell’s stability. Considered the master regulators of ion homeostasis, these integral membrane proteins enable the proper operation of the lysosome. Defects in the structure or function of these ion channels lead to the development of lysosomal storage diseases, neurodegenerative diseases and cancer. Although more than 50 years have passed since their discovery, lysosomes are not yet fully understood, with their ion channels being even less well characterized. However, significant improvements have been made in the development of drugs targeted against these ion channels as a means of combating diseases. In this review, we will examine how Ca²⁺, K⁺, Na⁺ and Cl⁻ ion channels affect the function of the lysosome, their involvement in hereditary and spontaneous diseases, and current ion channel-based therapies.     

Store-operated Ca-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca²⁺ store in smooth muscle triggered a Ca²⁺ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca²⁺ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca²⁺ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca²⁺ ions. Two activation mechanisms have been identified which involve Ca²⁺ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Ion-channels reconstituted into lipid bilayer from human sperm plasma membrane

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200 // trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na⁺-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about −18 mV, which is close to the predicated value of a perfect Nernst K⁺ or Na⁺ electrode. In 50//10 mM CaCl₂ solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about −20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction. Mol. Reprod. Dev. 50:354–360, 1998. © 1998 Wiley-Liss, Inc.     

Imaging of Calcium Channels During Polarity Induction in Plant Cells

Understanding the molecular basis of polarity induction in plant cells is a research aspect that extends from signal perception and transduction to morphogenesis. A gradient of cytoplasmic ion fluxes generated through ion channels plays a crucial role in subsequent events leading to polar growth. Convincing evidence is now available implicating temporal and spatial distribution of Ca²⁺ in cytoplasm, generated by localized activity of calcium channels, as the early biochemical events associated with polarity induction. Ion channel antagonists are common tools for studying ion channel structure and function. Coupled with a fluorescent dyes, calcium channel antagonists (phenylalkylamine and dihydropyridine), have been used to localize L-type calcium channels. Additionally, the advent of Confocal Laser Scanning Microscopy has made possible the visualization of Ca²⁺ channels in plant cells. Persisting problems of dye loading and their cellular compartmentation have been addressed by developing a variety of experimental protocols. Present article highlights the current state of our understanding of these concepts, methodologies and their applications in different aspects of plant development.     

KCa and Ca channels: The complex thought

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca^2 +-activated K⁺ channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca^2 + channels are divided into two main families, voltage gated/voltage dependent Ca^2 + channels and non-voltage gated/voltage independent Ca^2 + channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca^2 + channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca^2 +. Non-voltage gated Ca^2 + channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca^2 + channels while non-voltage gated Ca^2 + channels are activated by Ca^2 + depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca^2 + channel families also control constitutive Ca^2 + entries. For reducing the energy consumption and for the fine regulation of Ca^2 +, KCa and Ca^2 + channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa–Ca^2 + channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane¹. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex^2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein''s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (K_Na) channels by GPCRs.     

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca²⁺ and Mg²⁺ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.     

Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K _o ⁺ ] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K⁺ concentration. As well as changes in serum [K _o ⁺ ], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K _o ⁺ -dependent, and the suggestion has been made that pH and K _o ⁺ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K _o ⁺ . It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K⁺ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K⁺ channels in the open vs inactivated states of the channels by physiological H⁺ and K⁺ concentrations represents an important pathway to control Kv channel function in health and disease.     

On the other hand: The stereoselectivity of drug action at ion channels

Ion channels are pharmacological receptors with specific drug binding sites. These binding sites define specific structure–function relationships for the actions of drug classes. Interpretation of these structure–function relationships may be complex because of state-dependent drug-channel interactions. These state-dependent interactions determine affinity and access of drug to binding sites and may result in both quantitative and qualitative changes in structure–function relationships including stereoselectivity. A channel-active drug may exhibit antagonist or activator properties according to membrane potential and the stereoselectivity of interaction may also change with channel state. © 1994 Wiley-Liss, Inc.