Gating pore currents,a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na⁺ and Ca²⁺ channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K⁺ ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K⁺ channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K⁺ (K2P) channels also contribute to the steady outward leak of K⁺ ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K⁺ channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Identification of a Novel Bacterial K<Superscript>+</Superscript> Channel

In an attempt to explore unknown K⁺ channels in mammalian cells, especially ATP-sensitive K⁺ (K_ATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian K_ATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K⁺ channel current that was inwardly rectified and had a reversal potential close to K⁺ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific K_ATP channel openers or blockers did not alter the K⁺ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of K_ATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K⁺ currents were inhibited by protopine (a nonspecific K⁺ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K⁺ channel in mammalian cells.     

Functional properties of the epithelial Ca2+ channel, ECaC.

ECaC is the first member of a new subfamily of Ca2+ channels embedded in the large TRPC family that includes numerous channel proteins. The channel has been proposed as the main gatekeeper of transcellular Ca2+ transport in kidney and intestine. The functional characterization of this channel is evolving rapidly and may have far reaching consequences for other channels of the TRPC family. The goal of this mini-review is to summarize the major functional and structural characteristics of ECaC, including (i) its proposed functional role, (ii) its channel structure and expression pattern, (iii) its main electrophysiological characteristics and (iv) its regulation.     

Pathophysiology of voltage-gated K channels in vascular smooth muscle cells: Modulation by protein kinases

In this review, the pathological alteration and clinical relevance of voltage-gated K⁺ (Kv) channels and their specific regulation by protein kinase-dependent signaling in vascular smooth muscle cells are described, particularly focusing on the pulmonary vasculature. The physiological relevance, channel characteristics, pharmacological modulation, and expression of Kv channels vary between different arterial beds and between subdivisions of arteries within those vascular beds. Although detailed signaling cascades regulating Kv channels are not clearly elucidated, it is known that the Kv channels in vascular smooth muscle cells can be tightly regulated by protein kinases C (PKC) and A (PKA). Alterations in Kv channel expression and function has been noted in pathological and pathophysiological conditions including hypertension (pulmonary and systemic), in diabetes and in individuals subjected to prolonged hypoxia (high altitude living). Vascular Kv channels are potential therapeutic targets in diseases such as pulmonary arterial hypertension and, therefore, it is important to understand the specific pharmacological modulation of Kv channel isoforms in different vascular beds.     

Functional and biochemical evidence for G-protein-gated inwardly rectifying K+ (GIRK) channels composed of GIRK2 and GIRK3

G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.     

Physiological roles of NAADP-mediated Ca<Superscript>2+</Superscript> signaling

Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca²⁺ mobilizing messengers in that its principal function is to mobilize Ca²⁺ from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca²⁺ release channel distinct from the well characterized Ca²⁺ release channels on the (sarco)-endoplasmic reticulum (ER), inositol trisphosphate and ryanodine receptors. In this review, we discuss the emergence of a novel family of endolysosomal channels, the two-pore channels (TPCs), as likely targets for NAADP, and how molecular and pharmacological manipulation of these channels is enhancing our understanding of the physiological roles of NAADP as an intracellular Ca²⁺ mobilizing messenger.     

Distinct pharmacological profiles of ORAI1, ORAI2, and ORAI3 channels

The ubiquitous Ca²⁺ release-activated Ca²⁺ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd³⁺ (5μM), 2-APB (5 μM and 50 μM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.     

A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca²⁺. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, I_Cl.vol.     

The Role of the Golgi-Resident SPCA Ca<Superscript>2+</Superscript>/Mn<Superscript>2+</Superscript> Pump in Ionic Homeostasis and Neural Function

Recent evidence highlights the functional importance of the Golgi apparatus (GA) in neurological diseases. The functions of the mammalian GA, in addition to the processing and transport of cargo, also include ionic homeostasis. Besides Ca²⁺-release channels which serves GA as an agonist-sensitive intracellular Ca²⁺ store, and Ca²⁺-binding proteins, the GA contains Ca²⁺-uptake mechanisms consisting of the well-known sarco-endoplasmic reticulum Ca²⁺-transport ATPases and the much less characterized secretory-pathway Ca²⁺-transport ATPases (SPCA). SPCA can transport both Ca²⁺ and Mn²⁺ into the Golgi lumen and therefore is involved in the cytosolic and intra-Golgi Ca²⁺ and Mn²⁺ homeostasis. It has shown that both of the mRNA and protein of SPCAs are highly expressed in brain. In addition, brain is the region with the highest activity of SPCA isoforms, which may be related to the involvement of Ca²⁺ and Mn²⁺ homeostasis in neural functions. In this review, we compile some recent findings showing that the SPCA isoform plays a much more important role in intracellular ionic homeostasis than previously anticipated and illustrating the involvement of SPCA isoforms in certain neurophysiological or neuropathological process. We are interested in gaining insight into the intricate role of the SPCA pumps to explain the GA-specific functions in neurological disorders.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Two-pore domain potassium channels: variation on a structural theme

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na(+) and Ca(2+) channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K(+) ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K(+) channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K(+) (K2P) channels also contribute to the steady outward leak of K(+) ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K(+) channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.     

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.     

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2X(f)) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2X(f) was inhibited by suramin, PPADS and TNP-ATP. Activation of P2X(f) attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2X(f) plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2X(f) inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells.     

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

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.     

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.     

Extraction of K<Superscript>+</Superscript> Selective Channels from Excitable Tissue

ALTHOUGH the existence of ion conductance channels in excitable tissues is strongly suspected¹, there has been no way of measuring the occurrence of such channels in extracts of nerve or brain until the advent of lipid bilayer techniques², improved to the extent of allowing detection of a few channels³. The observation⁴ of ion channel formation in lipid bilayers by gramicidin A and its elucidation as a helical peptide structure^5,6 has prompted the search for substances of similar properties in extracts of nervous tissue. This is made more plausible by the fact that the channel conductance Δ(Na⁺) for the alkyldiamide derivatives of gramicidin is close to the value of 2 × 10^?10, which was estimated for nerve on pharmacological grounds⁷.