Homocysteine inhibits store-mediated calcium entry in human endothelial cells: Evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK_Ca channels opener NS1619 reversed thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; BK_Ca channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca²⁺ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK_Ca channels may play a regulatory role in it. (Mol Cell Biochem 269: 37–47, 2005)     

Different regulatory pathways involved in ATP-stimulated chloride secretion in rat epididymal epithelium

The regulatory pathways involved in the ATP-stimulated CI^? secretion across rat epididymal epithelium were investigated by the short-circuit current (I_SC) technique. Biphasic characteristic was observed in the I_SC responded to ATP (0.01-10 m?M). Inhibitor of P₁ receptor, 8-phenyltheophylline (up to 100 m?M), did not have any effect on both phases of the ATP-stimulated I_SC. The order of potency for stimulation of the two phases in I_SC was ATP>ADP> AMP, adenosine, consistent with the presence of P₂-purinoceptors. CI^? channel blocker, disulfonic acid stilbene (DIDS, 300 m?M), only inhibited the first peak of the ATP-stimulated I_SC while diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced both, indicating the involvement of different conductance pathways. DIDS was found to have an inhibitory effect on Ca²⁺-activated I_SC (induced by ionomycin, 10 m?M) but not cAMP-activated I_SC (induced by forskolin, 1 m?M) which could only be blocked by DPC. Both peaks of the ATP-activated I_SC could be significantly inhibited by pretreatment with a Ca²⁺-chelating agent, BAPTA-AM (50 m?M). An increase in cellular cAMP content upon stimulation of ATP was measured by radioimmunoassay. No significant increase in cAMP production was observed in cells stimulated with adenosine. The ATP-induced cAMP increase was prevented by pretreatment with BAPTA-AM (100 m?M) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca²⁺ concentration. These results indicate that the ATP-stimulated CI^? secretion could be mediated by Ca²⁺ and cAMP-dependent regulatory pathways giving rise to the biphasic nature of the ATP-induced I_SC. © 1995 Wiley-Liss, Inc.     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

Contribution of BKCa-Channel Activity in Human Cardiac Fibroblasts to Electrical Coupling of Cardiomyocytes-Fibroblasts

Cardiac fibroblasts are involved in the maintenance of myocardial tissue structure. However, little is known about ion currents in human cardiac fibroblasts. It has been recently reported that cardiac fibroblasts can interact electrically with cardiomyocytes through gap junctions. Ca²⁺-activated K⁺ currents (I _K[Ca]) of cultured human cardiac fibroblasts were characterized in this study. In whole-cell configuration, depolarizing pulses evoked I _K(Ca) in an outward rectification in these cells, the amplitude of which was suppressed by paxilline (1 μM) or iberiotoxin (200 nM). A large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channel with single-channel conductance of 162 ± 8 pS was also observed in human cardiac fibroblasts. Western blot analysis revealed the presence of α-subunit of BK_Ca channels. The dynamic Luo-Rudy model was applied to predict cell behavior during direct electrical coupling of cardiomyocytes and cardiac fibroblasts. In the simulation, electrically coupled cardiac fibroblasts also exhibited action potential; however, they were electrically inert with no gap-junctional coupling. The simulation predicts that changes in gap junction coupling conductance can influence the configuration of cardiac action potential and cardiomyocyte excitability. I _k(Ca) can be elicited by simulated action potential waveforms of cardiac fibroblasts when they are electrically coupled to cardiomyocytes. This study demonstrates that a BK_Ca channel is functionally expressed in human cardiac fibroblasts. The activity of these BK_Ca channels present in human cardiac fibroblasts may contribute to the functional activities of heart cells through transfer of electrical signals between these two cell types.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na⁺−H⁺ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH₄Cl) to the superfusate, or by changing the bath solution saturated with 10% CO₂+90% O₂ to a solution saturated with 100% O₂. Both approaches evoked hyperpolarization of EC. After intracellular Ca²⁺ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH₄Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH₄Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na⁺-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca²⁺-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca²⁺ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na⁺−H⁺-antiporter, inhibits influx of extracellular Ca²⁺ into EC under ATP stimulation.     

Differential Regulation of Action Potentials by Inactivating and Noninactivating BK Channels in Rat Adrenal Chromaffin Cells

Large-conductance Ca²⁺-activated K⁺ (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca²⁺ and membrane potential. In rat chromaffin cells (RCC), inactivating BK_i and noninactivating (BK_s) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK_i and BK_s current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK_i current facilitates repetitive firing of APs, whereas BK_s current does not. However, the key functional difference between BK_i and BK_s current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK_i current at a given [Ca²⁺].     

Differential segmental activation of Ca-dependent CIand Kchannels in pulmonary arterial myocytes

Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca²⁺-dependent CI⁻(CI_Ca) and K⁺(K_Ca) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca²⁺] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca²⁺oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of K_Caand CI_Caconductances. Resistance arteries contained a more homogeneous cell population with predominance of CI_Cachannel activation. The relation between K_Caand CI_Caconductances in a given conduit myocyte determines the size of the V_mchange in response to a rise of cytosolic [Ca²⁺]. Conduit myocytes tend to hyperpolarize towards the K⁺equilibrium potential ( − 90 m V). In resistance myocytes, release of Ca²⁺from stores activates CI_Cachannels and brings V_mto a value close to the chloride equilibrium potential ( − 20 or − 30 m V) thus favouring opening of Ca²⁺channels and Ca²⁺influx. In resistance vessels CI_Cachannels contribute to link agonist-induced Ca²⁺release from stores and membrane depolarization, thus permitting protracted vasoconstriction.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

The Simultaneous Measurement of Epithelial Ion Transport and Intracellular Free Ca2+ in Cultured Equine Sweat Gland Secretory Epithelium

We explored the relationship between nucleotide-evoked changes in intracellular free calcium ([Ca²⁺] _i ) and anion secretion by measuring [Ca²⁺] _i and I _SC simultaneously in Fura-2-loaded, cultured equine sweat gland epithelia. Apical ATP, UTP or UDP elicited sustained increases in [Ca²⁺] _i that were initiated by the mobilization of cytoplasmic Ca²⁺ but maintained by Ca²⁺ influx. However, although these nucleotides also increased I _SC , this response was transient whereas the [Ca²⁺] _i signals were sustained. Experiments in which external Ca²⁺ was removed/replaced showed that Ca²⁺ entering nucleotide-stimulated cells elicited very little change in I _SC . Cross desensitization experiments showed that UTP-stimulated epithelia became insensitive to ATP but that UTP could increase both [Ca²⁺] _i and I _SC in ATP-stimulated cells by activating `pyrimidinoceptors' essentially insensitive to ATP. Thapsigargin evoked a sustained rise in [Ca²⁺] _i that was accompanied by a maintained increase in I _SC . However, this increase in I _SC was dependent upon external Ca²⁺ and so the responses to nucleotides and thapsigargin have different properties. ATP increased I _SC in thapsigargin-treated cells without causing any rise in [Ca²⁺] _i while ionomycin increased both parameters. The data therefore show that apical P2Y receptors allow nucleotides to increase I _SC via two mechanisms, one of which appears to be [Ca²⁺] _i -independent control of anion channels. Received: 8 December 1998/Revised: 23 April 1999     

Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells

Our previous study demonstrated that a large-conductance Ca²⁺-activated K⁺ current (BK_Ca), a voltage-gated TTX-sensitive sodium current (I_Na.TTX), and an inward rectifier K⁺ current (I_Kir) were heterogeneously present in most of human cardiac c-kit⁺ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit⁺ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BK_Ca with paxilline, but not I_Na.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of I_Kir with Ba²⁺ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BK_Ca channels, but not I_Na.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit⁺ progenitor cells.     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Description and role in proliferation of iberiotoxin-sensitive currents in different human mammary epithelial normal and cancerous cells

Several studies suggested that potassium channels are involved in the proliferation of cancer cells but the involvement of the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in the cancerous phenomenon is still controversial. In the present study, we used iberiotoxin, a specific blocker of BK_Ca, and report the activity of an iberiotoxin-sensitive current in various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-435s) as well as in normal mammary epithelial cells (HME).Iberiotoxin and NS1619, an activator of BK_Ca, did not interfere with either cell proliferation or with the invasive properties of the cells, under normal culture conditions. However, extracellular pulses of ATP, which induced transient increases in intracellular Ca²⁺ concentration, revealed a significant reduction effect of iberiotoxin on cell proliferation.We conclude that the iberiotoxin-sensitive current is not involved in cell proliferation in basal conditions but participates when the intracellular Ca²⁺ concentration is increased. These experiments also suggest that BK_Ca channels are not involved in the cancerous transformation and are probably a relic from normal cells.     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Large conductance, Ca-activated K channels (BKCa) and arteriolar myogenic signaling

Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this vascular smooth muscle (VSM) response are events including membrane depolarization, Ca²⁺ entry and mobilization, and activation of contractile proteins. Large conductance, Ca²⁺-activated K⁺ channel (BK_Ca) has been implicated in several of these steps including, (1) channel closure causing membrane depolarization, and (2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BK_Ca activity (subunit composition, membrane potential (Em) and Ca²⁺ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity in BK_Ca channel activity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BK_Ca channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.     

Nonselective Cation Channels in Endothelial Cells Derived from Human Umbilical Vein

(i) We have used a combined patch-clamp and fura-2 fluorescence technique to characterize a nonselective cation channel (NSC) in Ea.hy926 (EA) cells, an endothelial cell line derived from human umbilical vein. (ii) Stimulation with ATP, histamine and bradykinin activated slowly and with a long delay after application of the agonist, a nonselective cation current (I _NSC) which is time- and voltage-independent. The permeability sequence for cations was P _Na > P _Cs >> P _NMDG , P _Ca . In the absence of external Ca²⁺ and at rather high concentrations, La³⁺ and Gd³⁺ blocked I _NSC . (iii) Single channel analysis revealed that ATP activates in the cell-attached configuration a nonselective cation channel with a conductance of approximately 24 pS and a permeation sequence identical to that of the macroscopic current. The channel activity disappeared after membrane excision. (iv) Activation of NSC required physiological intracellular Ca²⁺ levels (100 nm or higher). All agonists failed to activate NSC if cytosolic Ca²⁺ ([Ca²⁺] _i ) was lowered by 10 mm BAPTA. Clamping internal Ca²⁺ at 1 μm sometimes (8 out of 17 cells) spontaneously activated I _NSC in the absence of any additional stimulus. (v) Application of 2,5-di-tert-butylhydroquinone and internal perfusion of inositol 1,4,5-trisphosphate also activated I _NSC . The phospholipase C inhibitor, U-73122 inhibited I _NSC and the sustained Ca²⁺ plateau during agonist stimulation whereas the inactive analogue, U-73343 had no effect. (vi) These results indicate NSC may act as a Ca²⁺ entry pathway in endothelium. [Ca²⁺] _i and inositol 1,4,5-trisphosphate play a role in the activation cascade of NSC, and possibly also store depletion. Received: 13 October 1998/Revised: 28 January 1999     

On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Electrophysiology of human cardiac atrial and ventricular telocytes

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber‐specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole‐cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c‐Kit and PDGFR‐β were identified. These cells expressed large conductance Ca²⁺‐activated K⁺ current (BK_Ca) and inwardly rectifying K⁺ current (IK_ir), but not transient outward K⁺ current (I_to) and ATP‐sensitive potassium current (K_ATP). The active channels were functionally competent with demonstrated modulatory response to H₂S and transforming growth factor (TGF)‐β1 whereby H₂S significantly inhibited the stimulatory effect of TGF‐β1 on current density of both BK_Ca and IK_ir. Furthermore, H₂S attenuated TGF‐β1‐stimulated KCa1.1/Kv1.1 (encode BK_Ca) and Kir2.1 (encode IK_ir) expression in TCs. Our results show that functionally competent K⁺ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.