Sodium-permeable Channels Research Articles

Membrane potential changes occurring upon acidification influence the binding of small-molecule inhibitors to ASIC1a

Acid-sensing ion channels (ASICs) are proton-activated, sodium-permeable channels, highly expressed in both central and peripheral nervous systems. ASIC1a is the most abundant isoform in the central nervous system and is credited to be involved in several neurological disorders including stroke, multiple sclerosis, and epilepsy. Interestingly, the affinity of ASIC1a for two antagonists, diminazene and amiloride, has recently been proposed to be voltage sensitive. Based on this evidence, it is expected that the pharmacology of ASIC1cannot be properly characterized by single-cell voltage-clamp, an experimental condition in which membrane potential is maintained close to resting values. In particular, these measurements do not take into account the influence of the membrane potential depolarization induced by ASIC1a activation during acidosis or neuronal activity.We show here the voltage-dependence of some small molecules antagonists (diminazene, amiloride and a new patented drug from Merck), but not of Psalmotoxin 1, a peptide binding to regions other than the pore. We also demonstrate that the opening of ASIC1a induced by moderate acidosis determines a depolarization sufficient to change the affinity of small molecule antagonists. The characterization of this mechanism was performed on CHO-K1 expressing ASIC1a and further confirmed in hippocampal neurons in culture. Finally, perforated-patch experiments indicate that intracellular modulations do not play a role in the voltage-dependent binding of small molecules. Since ASIC1a activation promotes a membrane depolarization that may influence the binding of small molecules, we propose to adopt experimental methods that do not interfere with the membrane potential for the drug screening of ASIC1a modulators.

Regulation of Lung Epithelial Sodium Channels by Cytokines and Chemokines.

Acute lung injury leading to acute respiratory distress (ARDS) is a global health concern. ARDS patients have significant pulmonary inflammation leading to flooding of the pulmonary alveoli. This prevents normal gas exchange with consequent hypoxemia and causes mortality. A thin fluid layer in the alveoli is normal. The maintenance of this thin layer results from fluid movement out of the pulmonary capillaries into the alveolar interstitium driven by vascular hydrostatic pressure and then through alveolar tight junctions. This is then balanced by fluid reabsorption from the alveolar space mediated by transepithelial salt and water transport through alveolar cells. Reabsorption is a two-step process: first, sodium enters via sodium-permeable channels in the apical membranes of alveolar type 1 and 2 cells followed by active extrusion of sodium into the interstitium by the basolateral Na+, K+-ATPase. Anions follow the cationic charge gradient and water follows the salt-induced osmotic gradient. The proximate cause of alveolar flooding is the result of a failure to reabsorb sufficient salt and water or a failure of the tight junctions to prevent excessive movement of fluid from the interstitium to alveolar lumen. Cytokine- and chemokine-induced inflammation can have a particularly profound effect on lung sodium transport since they can alter both ion channel and barrier function. Cytokines and chemokines affect alveolar amiloride-sensitive epithelial sodium channels (ENaCs), which play a crucial role in sodium transport and fluid reabsorption in the lung. This review discusses the regulation of ENaC via local and systemic cytokines during inflammatory disease and the effect on lung fluid balance.

Cav3 T-type channels: regulators for gating, membrane expression, and cation selectivity

Cav3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Cav1 and Cav2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located "gating brake" works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III-IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I-II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Cav1 and Cav2 channels but also the sodium ion selectivity of Nav1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Cav3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Nav1 sodium channels in invertebrates. Highly sodium-permeant Cav3 channels can functionally replace Nav1 channels in species where they are lacking, such as in Caenorhabditis elegans.

The Contribution of Epithelial Sodium Channels to Alveolar Function in Health and Disease

Amiloride-sensitive epithelial sodium channels (ENaC) play an important role in lung sodium transport. Sodium transport is closely regulated to maintain an appropriate fluid layer on the alveolar surface. Both alveolar type I and II cells have several different sodium-permeable channels in their apical membranes that play a role in normal lung physiology and pathophysiology. In many epithelial tissues, ENaC is formed from three subunit proteins: alpha, beta, and gamma ENaC. Part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. Thus, lung epithelium has enormous flexibility to alter the magnitude of salt and water transport. In lung, ENaC is regulated by many transmitter and hormonal agents. Regulation depends upon the type of sodium channel but involves controlling the number of apical channels and/or the activity of individual channels.

Ions in smooth muscle, now and then

Edith Bülbring is widely regarded as a pioneer in the smooth muscle field – driving forward understanding of electrophysiology and inspiring the next generation of investigators. In September 2005 there was a meeting in Oxford to celebrate the contributions of two of these investigators – Tom Bolton and Alison Brading, who met in Oxford under Edith's tutorage. Although not publishing together they have been spirited contemporaries and provided us with almost the same number of original research papers each. Between them they have so far published 126 papers in The Journal of Physiology, work that has attracted 824 citations. Tom's most cited paper is on spontaneous transient outward currents in smooth-muscle cells (Benham & Bolton, 1986), while Alison's is one of her earliest papers on CoEDTA in smooth muscle (Brading & Jones, 1969). Tom and Alison are now household names in the field and remain active contributors. Mike Kotlikoff started off the scientific programme of the Meeting with a talk on ‘calcium indicator mice’, showing striking images of intracellular calcium measured in vivo. This was to set the scene for talks on various aspects of calcium signalling in smooth muscle by Dima Gordienko, Mark Evans, Yuji Imaizumi and Casey van Breemen. Coupled with this we saw studies exploring endogenous cationic channels and transient receptor potential channels by Sasha Zholos, William Large, Ryuji Inoue, Seiichi Komori, Chris Benham, Phil Aaronson and Bernd Nilius. Kenton Sanders gave the Smooth Muscle Special Interest Group Lecture, Diomedes Logothetis the Ion Channel Lecture – showing two extremes, from the origins of rhythmicity in the gut, to the molecular basis of PIP2 regulation of an array of membrane proteins including channels. The theme of physiological rhythmicity was taken up by Sue Wray, Mark Hollywood, Hikaru Suzuki and Tom Bolton. Steffen Hering gave us an intriguing overview of the still-unsolved mechanism of action of calcium antagonists, while there were also new insights into transcriptional control mechanisms, by Karen Lounsbury and David Beech, and insightful talks on the functions, complexities and diversity of potassium channels by Rick Lang, Alison Gurney and Ligia Toro. In this themed edition of The Journal of Physiology we are fortunate to have a series of excellent and concise reviews from established as well as up-and-coming investigators in the field. The reviews provide a flavour and formal record of the meeting and also a glimpse into the continuing vibrancy of the field, recent advances, and questions remaining unsolved. Brading (2006), McHale et al. (2006), Noble et al. (2006) and Sanders & Koh (2006) focus on mechanisms of rhythmicity in various visceral organs. Underlying platforms of calcium-release events and calcium-activated channels predominate, while Sanders & Koh (2006) expand on their hypothesis for the role of K2P potassium channels in periods of quiescence. There is also the topical thread of interstitial cells and their integration in smooth muscle tissues. Bolton (2006) provides an elegant commentary on elementary calcium-release events, while Albert & Large (2006) develop hypotheses for second messenger pathways regulating calcium- and sodium-permeable channels. Aaronson et al. (2006) provide a thoughtful review of mechanisms underlying hypoxic pulmonary vasoconstriction, a subject that has consumed the attention of many investigators in the smooth muscle field and yet retained significant unanswered questions and areas of controversy. Barlow et al. (2006) and Lu et al. (2006) provide a distinctly molecular perspective, poised in physiological contexts. Barlow et al. (2006) pioneer studies of the link between smooth muscle excitation and transcriptional activity, emphasizing the importance of calcium entry for gene expression as well as regulation of contraction and electrical rhythmicity. Lu et al. (2006) will have especially broad appeal, providing an important and surprisingly rare summary of the protein partners and regulation of the BKCa potassium channel – an old favourite in smooth muscle, but also major player in other cell types. It is hoped that the selection of reviews will provide a useful introduction for those outside the field and raise challenging questions for those already actively engaged or considering joining. Humans and many other animals would be lost without their many tubes and reservoirs, precisely regulated by the smooth muscle cell in its many guises. Despite profound advances in our understanding of these cells key questions remain unanswered and enormous facets of their origin, survival, phenotype and behaviour are mysterious or simply completely unexplored.

Highlights from the Literature

Highlights from the Literature

Regulation of Na+ Channels in Lung Alveolar Type II Epithelial Cells

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and pathologic states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins designated alpha-ENaC, beta-ENaC, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this article, we discuss the regulation of ENaCs composed of varying subunits and some of the implications of the regulation for normal pulmonary function.

Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells.

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport, with the role of alveolar type I cells being less clear. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and in pathological states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins, designated alpha-, beta-, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from the assembling of different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung, which has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this review, we discuss the biophysical properties and occurrence of these various channels and some of the mechanisms for their regulation.

Stressed plants need their vitamins

With the need to irrigate more and more land to provide food for the growing human population, the design of crops that have increased salt resistance is becoming a crucial issue. A variety of approaches have been used to dissect the mechanisms of plant tolerance to salt stress. Expression of plant genes in yeast has enabled the discovery of HKT1, a sodium-coupled potassium transporter from wheat that constitutes one of the sodium entry pathways in plants. Electrophysiological studies of plant roots have uncovered the existence of non-selective sodium-permeable channels that could provide a pathway for sodium uptake into roots in several different species. More recently, the availability of the whole sequence of the Arabidopsis genome led to the discovery of NHX vacuolar sodium-proton antiporter genes, which improve plant resistance to salt stress by increasing sodium sequestration into the vacuole. One of the most powerful strategies to identify determinants of salt tolerance is a forward genetic approach. Initial screens for salt overly sensitive (sos) mutants identified three genes: SOS1, a putative sodium transporter, which points again to the importance of the control of solute transport through the membrane; and SOS2 and SOS3, which reveal the implication of calcium and kinase signaling during salt stress.

Disruption of actin filaments increases the activity of sodium-conducting channels in human myeloid leukemia cells.

With the use of the patch clamp technique, the role of cytoskeleton in the regulation of ion channels in plasma membrane of leukemic K562 cells was examined. Single-channel measurements have indicated that disruption of actin filaments with cytochalasin D (CD) resulted in a considerable increase of the activity of non-voltage-gated sodium-permeable channels of 12 pS unitary conductance. Background activity of these channels was low; open probability (po) did not exceed 0.01-0.02. After CD, po grew at least 10-20 times. Cell-attached and whole-cell recordings showed that activation of sodium channels was elicited within 1-3 min after the addition of 10-20 micrograms/ml CD to the bath extracellular solution or in the presence of 5 micrograms/ml CD in the intracellular pipette solution. Preincubation of K562 cells with CD during 1 h also increased drastically the activity of 12 pS sodium channels. Whole-cell measurements confirmed that CD-activated channels were permeable to monovalent cations (preferentially to Na+ and Li+), but not to bivalent cations (Ca2+, Ba2+). Colchicine (1 microM), which affect microtubules, did not alter background channel activity. Our data indicate that actin filaments organization plays an important role in the regulation of sodium-permeable channels which may participate in providing passive Na+ influx in red blood cells.

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels.

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.

Sodium-selective channels in membranes of rat macrophages.

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of -60 to +40 mV. The channels had a unitary conductance of 10.2 +/- 0.2 pS with 145 mM Na+ in the external solution at 23-24 degrees C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na+ over K+ (as for Cs+, NH4+, Ca2+, Ba2+) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 +/- 0.2 pS in the Li(+)-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01-1 microM). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1-2 mM) and its derivative ethylisopropilamiloride (0.01-0.1 mM). The mechanism of channel block by amiloride and its analogue seems to be different.

Sodium-permeable channels in the apical membrane of human nasal epithelial cells

We used patch-clamp techniques to study the channels that underlie the Na+ conductance of the apical membrane of human normal nasal epithelial cells. Cells were cultured on permeable supports and studied after confluence. In 172 of 334 (52%) excised membrane patches, we observed 20-pS Na(+)-permeable channels that do not discriminate between Na+ and K+ (pNa/pK = 1.33). These nonselective cation channels contained subpopulations that differed by dependence of open probability on voltage and bath Ca2+ activity, suggesting two or more channel types with similar electrical properties. In the presence of 10(-4) M amiloride in the pipette, the proportion of excised patches with nonselective cation channels decreased to 52 of 139 patches (37%), but the decrease was spread across all subpopulations of nonselective cation channels in excised patches. Thus no distinctive Na(+)-selective amiloride-sensitive channels were identified in excised patches. In cell-attached patches, Na(+)-permeable channels were recorded in 56 of 262 patches (21%). Their conductance was 21.4 +/- 1.5 pS (n = 25), and most were selective for Na+ over K+ (pNa/pK > 6). In the presence of amiloride (10(-4) M) in the pipette, the frequency of lambda Na(+)-permeable channels in cell-attached patches decreased to 8 of 134 patches (6%), revealing a population of Na(+)-selective channels recorded in cell-attached patches that was inhibited by amiloride. We conclude that, in excised patches, Na(+)-permeable channels are nonselective for Na+ over K+ and < 30% appear to be amiloride sensitive. In contrast, in cell-attached patches, most channels that conduct sodium are 1) selective for Na+ over K+ and 2) amiloride sensitive. Although we have not discovered the explanation for the discrepancy between cell-attached and excised patch data, we speculate that the channels recognized on cell account for the amiloride-sensitive Na+ conductance of the apical membrane, whereas the excision process alters the properties of the Na(+)-permeable channels and/or activate new channels.

Sodium dependence of NMDA's effects on cyclic GMP production in immature rat cerebellar slices

In immature rat cerebellar slices, the calcium-dependent increase in cyclic GMP levels provoked by N- methyl- d-aspartic acid (NMDA) (80 μM) displayed sodium dependence using bis-(2-hydroxyethyl)-dimethyl ammonium chloride, N-methyl-glucamine or Tris as sodium substitutes. The effects of NMDA (and also of veratrine, 100 μM) were attenuated by substitution of sodium chloride by lithium chloride. The response produced by depolarization with KCI (50 mM) was not affected by lithium substitution. As lithium is believed to permeate sodium-permeable channels but is not a substrate for sodium/calcium exchange, the data suggest that calcium entry mediated by the reverse mode of sodium/calcium exchange may play a contributory role to the calcium entry provoked by NMDA and veratrine.