Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability

The present study evaluated the necessity of store-operated Ca(2+) entry in mediating thrombin-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 microM) activated Ca(2+) entry through a common pathway in confluent BPAECs. Similar increases in MLC(20) phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC(20) phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC(20) phosphorylation when Ca(2+) influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 A) transfer across confluent BPAEC monolayers. Activation of store-operated Ca(2+) entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca(2+) entry is coupled with MLC(20) phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca(2+) entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca(2+) signaling.     

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.     

Differential expression of voltage-gated Ca(2+)-currents in cultivated aortic myocytes.

The expression of different types of Ca(2+)-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca(2+)-currents were identified as either low- or high voltage-activated (ICa,LVA or ICa,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca(2+)-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus, both LVA and HVA Ca(2+)-current types contribute to Ca(2+)-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.     

Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells

The TRPC1 (transient receptor potential canonical-1) channel is a constituent of the nonselective cation channel that mediates Ca2+ entry through store-operated channels (SOCs) in human endothelial cells. We investigated the role of protein kinase Calpha (PKCalpha) phosphorylation of TRPC1 in regulating the opening of SOCs. Thrombin or thapsigargin added to the external medium activated Ca2+ entry after Ca2+ store depletion, which we monitored by changes in cellular Fura 2 fluorescence. Internal application of the metabolism-resistant analog of inositol 1,4,5-trisphosphate (IP3) activated an inward cationic current within 1 min, which we recorded using the whole cell patch clamp technique. La3+ or Gd3+ abolished the current, consistent with the known properties of SOCs. Pharmacological (G?6976) or genetic (kinase-defective mutant) inhibition of PKCalpha markedly inhibited IP3-induced activation of the current. Thrombin or thapsigargin also activated La3+-sensitive Ca2+ entry in a PKCalpha-dependent manner. We determined the effects of a specific antibody directed against an extracellular epitope of TRPC1 to address the functional importance of TRPC1. External application of the antibody blocked thrombin- or IP3-induced Ca2+ entry. In addition, we showed that addithrombin or thapsigargin induced phosphorylation of TRPC1 within 1 min. Thrombin failed to induce TRPC1 phosphorylation in the absence of PKCalpha activation. Phosphorylation of TRPC1 and the resulting Ca2+ entry were essential for the increase in permeability induced by thrombin in confluent endothelial monolayers. These results demonstrate that PKCalpha phosphorylation of TRPC1 is an important determinant of Ca2+ entry in human endothelial cells.     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9- anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.

Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.     

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.     

Selective downregulation of the BKbeta1 subunit in diabetic arteriolar myocytes

Diabetic retinopathy is an important cause of visual loss. Functional abnormalities including vasoconstriction precede structural changes. Using the streptozotocin-model of diabetes in rats, we have identified downregulation of the beta1 subunit of the BK channel in arteriole myocytes as a possible molecular mechanism underlying these early changes. BKbeta1 mRNA levels were reduced as early as one month after induction of diabetes, and BK Ca(2+)-sensitivity and caffeine-evoked BK currents were reduced at three months. This effect appears to be selective for the regulatory subunit, as BKalpha subunit expression was not altered at the mRNA level, and voltage-activated BK currents were unaltered. No changes were seen in voltage activated Ca(2+)-current, Ca(2+)-activated Cl(-)current, or A-type voltage activated K(+)-currents. Reduced Ca(2+)-activated BK activity may promote depolarization, Ca(2+)-channel activation and increased contraction under resting conditions or in response to Ca(2+)-mobilizing agonists.     

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.     

Store-operated cationic channels in human myeloid leukaemia K562 cells

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.     

Tumor necrosis factor-alpha-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability

We determined the effects of TNF-alpha on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-alpha exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the Ca(2+) store depletion-mediated Ca(2+) influx in response to thrombin exposure. We observed that thrombin-induced Ca(2+) influx in TNF-alpha-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated Ca(2+) influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombin-induced store Ca(2+) depletion in these cells caused approximately twofold greater increase in Ca(2+) influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPC1-transfected cells compared with control. To address the role of Ca(2+) influx via TRPC1 in signaling endothelial permeability, we measured actin-stress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF-alpha-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPC1-overexpressing HMEC compared with control cells. TNF-alpha-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of Ca(2+) influx and signaling of the increase in endothelial permeability.     

Divalent copper is a potent extracellular blocker for TRPM2 channel

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cationic channel in the TRP channel family. The channel activity can be regulated by reactive oxygen species (ROS) and cellular acidification, which has been implicated to the pathogenesis of diabetes and some neuronal disorders. However, little is known about the effect of redox-active metal ions, such as copper, on TRPM2 channels. Here we investigated the effect of divalent copper on TRPM2. TRPM2 channel was over-expressed in HEK-293 cells and the whole-cell current was recorded by patch clamp. We found the whole-cell current evoked by intracellular ADP-ribose was potently inhibited by Cu(2+) with a half maximal inhibitory concentration (IC(50)) of 2.59 μM. The inhibitory effect was irreversible. The single channel activity was abolished in the outside-out patches, and intracellular application of Cu(2+) did not prevent the channel activation, suggesting that the action site of Cu(2+) is located in the extracellular domains of the channel. TRPM2 current was also blocked by Hg(2+), Pb(2+), Fe(2+) and Se(2+). We concluded that Cu(2+) is a potent TRPM2 channel blocker. The sensitivity of TRPM2 channel to heavy metal ions could be a new mechanism for the pathogenesis of some metal ion-related diseases.     

Studies on the mechanism of thrombin. Interaction with fibrin

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.9 X 10(5) M-1. Ca2+ ions caused rapid desorption and elution of thrombin from fibrin monomer, and the Ca2+ concentration needed for maximal desorption depended on the fibrin:thrombin ratio. Mg2+, Mn2+, and Sr2+ also released thrombin from fibrin monomer but not as efficiently as Ca2+. These results indicate that divalent metal ions induce a physical change in fibrin monomer which results in desorption of thrombin. Thrombin binding to fibrin in a gel was compared to binding to fibrin monomer. These studies showed that as fibrin monomers polymerize to form the gel network, thrombin is released. Under static conditions the released thrombin remains associated with the gel because diffusion is limited by the gel. However, the thrombin can be readily removed when buffer is allowed to flow through the gel. These results lead to the possibility that thrombin binding to fibrin monomer and its subsequent release, either by Ca2+ or by polymerization, may have important consequences for regulating the effective thrombin concentration in vivo.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Endogenous cytosolic Ca(2+) buffering is necessary for TRPM4 activity in cerebral artery smooth muscle cells

The melastatin transient receptor potential (TRP) channel, TRPM4, is a critical regulator of smooth muscle membrane potential and arterial tone. Activation of the channel is Ca(2+)-dependent, but prolonged exposures to high global Ca(2+) causes rapid inactivation under conventional whole-cell patch clamp conditions. Using amphotericin B perforated whole cell patch clamp electrophysiology, which minimally disrupts cytosolic Ca(2+) dynamics, we recently showed that Ca(2+) released from 1,2,5-triphosphate receptors (IP(3)R) on the sarcoplasmic reticulum (SR) activates TRPM4 channels, producing sustained transient inward cation currents (TICCs). Thus, Ca(2+)-dependent inactivation of TRPM4 may not be inherent to the channel itself but rather is a result of the recording conditions. We hypothesized that under conventional whole-cell configurations, loss of intrinsic cytosolic Ca(2+) buffering following cell dialysis contributes to inactivation of TRPM4 channels. With the inclusion of the Ca(2+) buffers ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 10mM) or bis-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 0.1mM) in the pipette solution, we mimic endogenous Ca(2+) buffering and record novel, sustained whole-cell TICC activity from freshly-isolated cerebral artery myocytes. Biophysical properties of TICCs recorded under perforated and whole-cell patch clamp were nearly identical. Furthermore, whole-cell TICC activity was reduced by the selective TRPM4 inhibitor, 9-phenanthrol, and by siRNA-mediated knockdown of TRPM4. When a higher concentration (10mM) of BAPTA was included in the pipette solution, TICC activity was disrupted, suggesting that TRPM4 channels on the plasma membrane and IP(3)R on the SR are closely opposed but not physically coupled, and that endogenous Ca(2+) buffer proteins play a critical role in maintaining TRPM4 channel activity in native cerebral artery smooth muscle cells.     

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.