A mechanosensitive ion channel in Schizosaccharomyces pombe.

Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique. In addition to several voltage-gated ion channels, we encountered the activities of a mechanosensitive ion channel with a conductance of 180 pS. Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuration. This channel opens at pressures of cm Hg applied to whole blebs and it passes cations, including Ca2+. It is inactivated by membrane depolarizations and blocked by Gd3+. We discuss the possible functions of such a channel, including its activation upon cell cycle dependent cytoskeletal reorganizations.     

Mechanosensitive ion channels

The article concentrates on the concepts of mechanosensitive ion channels that are present in practically all cells of an organism. Considered are kinetic scheme and activation principles of mechanic-sensitive ion channels. The forces affecting those channels are discussed in detail. The qualities of the channels in lipid monolayer, bilayer and real cell membrane are under consideration. Discussed are various models that analyze possibilities of channel opening depending on the membrane tension. Under discussion are the data received from studying single channels, currents in whole-cell configuration and cloned channels built into bilayer, liposomes and membrane blebs. Problems of transmitting mechanic energy to the channel through the bilayer and through the cytoskeleton are investigated. Inhibitors and activators of mechanosensitive ion channels are mentioned and their effects are considered. The functional classification of mechanosensitive ion channels is given. Described are cation SACs, potassium SACs, Ca(2+)-sensitive and Ca(2+)-insensitive SACs, anion SACs, nonselective SACs and SICs. It is proved that mechanosensitive ion channels can produce considerable currents enough to change the cell electrogenesis.     

Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication

Tendon and other connective tissue cells are subjected to diverse mechanical loads during daily activities. Thus, fluid flow, strain, shear and combinations of these stimuli activate mechanotransduction pathways that modulate tissue maintenance, repair and pathology. Early mechanotransduction events include calcium (Ca2+) signaling and intercellular communication. These responses are mediated through multiple mechanisms involving stretch-activated channels, voltage-activated channels such as Ca(v)1, purinoceptors, adrenoceptors, ryanodine receptor-mediated Ca2+ release, gap junctions and connexin hemichannels. Calcium, diacylglycerol, inositol (1,4,5)-trisphosphate, nucleotides and nucleosides play intracellular and/or extracellular signaling roles in these pathways. In addition, responses to mechanical loads in tendon cells vary among species, tendon type, anatomic location, loading conditions and other factors. This review includes a synopsis of the immediate responses to mechanical loading in connective tissue cells, particularly tenocytes. These responses involve Ca2+ signaling, gap junctions and intercellular communication.     

Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha1A-containing high-voltage-activated calcium channels

We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.     

Functional properties and modulation of extracellular epitope-tagged Ca(V)2.1 voltage-gated calcium channels

Depolarisation-induced Ca2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca2+ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope-tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope-tagged Ca(V)2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca(V)2.1 alpha(1) subunit, which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising alpha(1) subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescence studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca(V)2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artificial sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca2+ channel for their selective modulation.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Beta1-integrins co-localize with Na,K-ATPase,epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes

Interactions between chondrocytes and their extracellular matrix are partly mediated by beta1-integrin receptors. Recent studies have shown that beta1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of beta1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The alpha subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using well-characterized antibodies. Indirect immunofluorescence revealed abundant expression of beta1-integrins and each of the selected systems in limb-bud chondrocytes. Two-fluorochrome immunostaining demonstrated that beta1-integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.     

Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers

Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21 pS and those activated by brevetoxin, 16 pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies.     

Association of Catsper1 or -2 with Ca(v)3.3 leads to suppression of T-type calcium channel activity

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.     

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.     

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin

The mechanism of action of gabapentin is still not well understood. It binds to the alpha(2)delta-1 and alpha(2)delta-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons.(1) In that study we only examined a 40-hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from Ca(V)2.1/beta(4)/alpha(2)delta-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by alpha(2)delta subunits, being prevented by mutations in either alpha(2)delta-1 or alpha(2)delta-2 that abolish gabapentin binding.(1) Furthermore, we also showed that the trafficking of alpha(2)delta-2 and Ca(V)2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of Ca(V)2.1 is not reduced by chronic gabapentin if it is co-expressed with alpha(2)delta-2 containing a point mutation (R282A) that prevents gabapentin binding.     

Voltage-gated calcium channels

The article concentrates on representatives of voltage-gated calcium ion channels that are present in practically all cells. Regarded is the molecular arrangement of a voltage-gated calcium channel that consists of pore forming trans-membrane alpha1 subunit and auxiliary alpha2delta-, beta-, and gamma-subunits. Under discussion are the structure and functions of each subunit. The principles of subunits interaction are considered. The research represents modern classification of voltage-gated calcium channels, draws parallels with the earlier classifications and discusses calcium currents going through various calcium channels. Considered are the problems of regulating the activity of voltage-gated channels by proteinkinases. The issues of blockers and activators of voltage-gated calcium channels are brought up. The article gives a detailed analysis of the mechanisms of voltage-gated calcium channels selectivity. The molecular organization of the selectivity filter is considered. Presented are the basic theories of permeability of voltage-gated calcium channels.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.