Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 µM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA).

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A(2) (PLA(2)) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca(2+)-activated chloride current (I(Cl(Ca))) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I(Cl(Ca)) in a concentration-dependent manner (IC(50)=4.2 µM, n(Hill)=1.1), without affecting the L-type Ca(2+) current. Unlike ACA, the non-selective PLA(2) inhibitor bromophenacyl bromide (BPB; 50 µM) had no effect on I(Cl(Ca)). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 µM) also had no effect on I(Cl(Ca)), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 µM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS;100 µM). Besides I(Cl(Ca)), ACA (50 µM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I(Cl(Ca)) are PLA(2)-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.

Inhibition of the magnesium-sensitive TRPM7-like channel in cardiac myocytes by nonhydrolysable GTP analogs: involvement of phosphoinositide metabolism.

BACKGROUND/AIMS: A magnesium-inhibited, transient receptor potential melastatin 7 (TRPM7)-like channel is expressed in cardiac cell membranes. The role and regulation of this channel by intracellular nucleotides and membrane components remain unclear. METHODS: We used the whole-cell voltage-clamp technique in pig isolated ventricular myocytes to investigate the effect of non-hydrolysable guanine nucleotides. RESULTS: The TRPM7-like current, induced by intracellular dialysis with low [Mg(2+)], remained stable when the intracellular solution contained GTP. Substituting GTP by GTP-gamma-S or Gp-pNp, but not GDP-beta-S, induced a run-down of the current. Under dialysis with GTP-gamma-S, inhibiting phospholipase C by edelfosine or intracellularly adding exogenous phosphatidylinositol-4,5-bisphosphate (PIP(2)) decreased run-down, whereas extracellularly applying carbachol and phenylephrine accelerated it. Pretreatment of cells with pertussis toxin did not prevent the run-down induced by GTP-gamma-S. CONCLUSION: Guanine nucleotides can modulate cardiac TRPM7-like channels via a mechanism linked to G proteins and to PIP(2) metabolism.

Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions.

PURPOSE: Thrombin, a serine protease, breaks down the barrier integrity of corneal endothelial cells by phosphorylation of the regulatory light chain of myosin II (myosin light chain; MLC), which induces contractility of the actin cytoskeleton. This study was undertaken to investigate the effect of thrombin on gap junctional (GJIC) and paracrine (PIC) intercellular communication in cultured bovine corneal endothelial cells (BCECs). METHODS: An intercellular Ca(2+) wave, a form of cell-cell communication, was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were imaged by fluorescence microscopy with a fluorescent calcium indicator, and the images were used to calculate the area reached by the Ca(2+) wave (active area). GJIC was assessed by fluorescence recovery after photobleaching (FRAP). Activity of hemichannels was assayed by lucifer yellow (LY) uptake and also by adenosine triphosphate (ATP) release by using the luciferin-luciferase technique. RESULTS: RT-PCR showed transcripts for PAR-1 and -2 receptors, but not for PAR-4 receptors. Immunocytochemistry showed thrombin-sensitive PAR receptors as well as trypsin-sensitive PAR-2 receptors. Both thrombin and the selective PAR-1 agonist TRAP-6 reduced the active area of the Ca(2+) wave. These agents also reduced the fluorescence recovery in FRAP experiments. The effect of thrombin on the Ca(2+) wave was inhibited by a peptide antagonist of PAR-1, but not by a PAR-4 antagonist. Pretreatment with ML-7 (an MLCK inhibitor), Y-27632 (a Rho kinase inhibitor) or chelerythrine (a PKC inhibitor) prevented the effect of thrombin on the Ca(2+) wave. Activation of PAR-1 did not affect the Ca(2+) wave propagation in cells pretreated with Gap26, which blocks hemichannels. However, PAR-1 activation decreased the active area in cells pretreated with Gap27, which inhibits gap junctions. Thrombin abolished enhancement of the Ca(2+) wave propagation by ARL-67156 (inhibitor of ecto-ATPases). The effect of the PAR-1 agonists on the Ca(2+) wave was not detectable in cells pretreated with exogenous apyrases. CONCLUSIONS: Thrombin inhibits intercellular Ca(2+) wave propagation in BCECs. This effect is due to activation of PAR-1 receptors and involves MLC phosphorylation by MLCK-, PKC- and Rho kinase-sensitive pathways. Thrombin mainly inhibits the ATP-mediated PIC pathway, and also reduces GJIC to a lesser extent.

Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells.

PURPOSE: In corneal endothelial cells, intercellular Ca(2+) waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercellular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells. METHODS: An intercellular Ca(2+) wave was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. The area of Ca(2+) wave propagation was measured by [Ca(2+)](i) imaging using the fluorescent dye Fluo-4. Gap junctional intercellular communication was assessed by fluorescence recovery after photobleaching. Activity of hemichannels was determined by uptake of the hydrophilic dye Lucifer yellow in a Ca(2+)-free medium containing 2 mM EGTA. Adenosine triphosphate (ATP) release in response to mechanical stimulation was measured using the luciferin-luciferase technique. Gap26, a connexin mimetic peptide, was used to block hemichannels. RESULTS: Exposure to thrombin or TRAP-6 (a selective PAR-1 agonist) inhibited the Ca(2+) wave propagation by 70%. Pretreatment with adenosine prevented this inhibitory effect of thrombin. NECA (a potent A2B agonist) and forskolin, agents known to elevate cAMP in bovine corneal endothelial cells, also suppressed the effect of thrombin. The A1 receptor agonist CPA failed to inhibit the effect of thrombin. Similar to the effects on Ca(2+) wave propagation, adenosine prevented the thrombin-induced reduction in the fluorescence recovery during photobleaching experiments. Furthermore, pretreatment with adenosine prevented both thrombin and TRAP-6 from blocking the uptake of Lucifer yellow in a Ca(2+)-free medium. However, adenosine was ineffective in overcoming the Gap26-mediated block of Lucifer yellow uptake. In consistence with Lucifer yellow uptake through hemichannels, the thrombin-induced inhibition of ATP release was overcome by pretreatment with adenosine. CONCLUSIONS: Adenosine prevents thrombin-induced inhibition of hemichannel-mediated paracrine intercellular communication and of gap junctional intercellular communication. The mechanism involves an increase in cAMP, which results in inhibition of RhoA and a subsequent decrease in myosin light chain phosphorylation. Since myosin light chain dephosphorylation causes a decrease in contractility of the actin cytoskeleton, the results suggest possible effects of the actin cytoskeleton on gap junctions and connexin hemichannels.

Novel mutation in the Per-Arnt-Sim domain of KCNH2 causes a malignant form of long-QT syndrome.

BACKGROUND: It has been proposed that the highest risk for cardiac events in patients with long-QT syndrome subtype 2 (LQT2) is related to mutations in the pore region of the KCNH2 channel. It has also been suggested that a subpopulation of LQT2 patients may benefit from pharmacological therapy with modified KCNH2 channel-blocking drugs. METHODS AND RESULTS: In a large LQT2 family (n=33), we have identified a novel nonpore missense mutation (K28E) in the Per-Arnt-Sim (PAS) domain of the KCNH2 channel associated with a malignant phenotype: One third of the suspected gene carriers experienced a major cardiac event. Wild-type and K28E-KCNH2 channels were transiently transfected in HEK293 cells. For the mutant channel, whole-cell patch-clamp analysis showed a reduced current density, a negative shift of voltage-dependent channel availability, and an increased rate of deactivation. Western blot analysis and confocal imaging revealed a trafficking deficiency for the mutant channel that could be rescued by the K+ channel blocker E-4031. In cells containing both wild-type and mutant channels, deactivation kinetics were normal. In these cells, reduced current density was restored with E-4031. CONCLUSIONS: Our data suggest that besides pore mutations, mutations in the PAS domain may also exhibit a malignant outcome. Pharmacological restoration of current density is promising as a mutation-specific therapy for patients carrying this trafficking-defective mutant.

ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells.

PURPOSE: Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs). METHODS: A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence. RESULTS: BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 +/- 3,200 microm2 (N=21). The percentage of responsive cells (defined as cells showing maximum NF >1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 microM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 microM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 microM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs. CONCLUSIONS: PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell-cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.

ATP release through connexin hemichannels in corneal endothelial cells.

PURPOSE: Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs). METHODS: A Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence. RESULTS: Flufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+). CONCLUSIONS: Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system.

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.

Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in cell culture.

PURPOSE: Mechanical stimulation induces intercellular Ca(2 +) waves in the corneal endothelium. The extent of the wave propagation is dependent on the activity of gap junctions, hemichannels, and ectonucleotidases. To further establish the use of a cell culture model to investigate intercellular communication, in this study, we have characterized the changes in the Ca(2 +) wave propagation in bovine corneal endothelial cells with prolonged time in culture. MATERIALS AND METHODS: Freshly isolated BCEC were cultured for a short term (8 to 14 days; referred to as "short term") and a long term (21 to 30 days; referred to as "long term"). Cell surface area and size were measured by confocal microscopy and flow cytometry, respectively. Calcium wave propagation was assayed by imaging spread of the Ca(2 +) waves elicited by mechanical stimulation. ATP release was assayed using Luciferin-Luciferase bioluminescence technique. RESULTS: Cells cultured for a long term showed larger surface area and size compared to those cultured for a short term, but a reduced spread of the Ca(2 +) wave. Exposure to exogenous apyrases, which can rapidly hydrolyze extracellular ATP, reduced the spread of the Ca(2 +) wave in both groups. The fractional decrease, however, was smaller in cells cultured for a long term. Exposure to ARL-67156 to inhibit the ectonucleotidases led to a larger enhancement of the active area in cells cultured for a long term. However, the active areas of the two groups were not significantly different in the presence of the drug. Furthermore, ATP release in response to mechanical stimulation was lower in cells cultured for a long term in the absence of ARL-67156 but not in its presence. CONCLUSIONS: BCEC cultured for a long term show an increase in cell surface area and cell size similar to the effect of aging in human corneas. Moreover, the cells cultured for a long term showed a reduced ATP-dependent paracrine intercellular communication, largely due to an increase in the activity of the ectonucleotidases.

The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.

PURPOSE: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. METHODS: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. RESULTS: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. CONCLUSIONS: Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

Gap junctional intercellular communication in bovine corneal endothelial cells.

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4. The area reached by the Ca(2+) wave, called the active area (AA), was determined as a measure of efficacy of IC. RT-PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 microM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca(2+) wave induced by point mechanical stimulation from 73,689 microm(2) to 26,936 microm(2), implying that GJIC contribution to the spread of the wave is at least approximately 63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5U/ml each; exogenous ATPases), suramin (200 microM; P2Y antagonist), or Gap26 (300 microM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than approximately 9% towards the active area of the Ca(2+) wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 microM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca(2+)](o)-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca(2+) wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the two pathways interact.

ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes.

The Mg(2+)-inhibited cation (MIC) current (I(MIC)) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg(2+) homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I(MIC), studied in the presence or absence of extracellular divalent cations, was sustained for >or=50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I(MIC) exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I(MIC) rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 microM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by prevention of its resynthesis (10 microM wortmannin or 15 microM phenylarsine oxide) induced rundown of I(MIC). Finally, loading ATP-depleted cells with exogenous PIP(2) (10 microM) prevented rundown. These results suggest that PIP(2), likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity.

Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation.

Cardiac tissue expresses several TRP proteins as well as a Mg2+ -inhibited, non-selective cation current (IMIC) that bears many characteristics of TRP channel currents. We used the whole-cell voltage clamp technique in pig and rat ventricular myocytes to characterize the permeation, blockage properties and regulation of the cardiac IMIC channels in order to compare them with TRP channels, in particular with Mg2+ -sensitive TRPM6 and TRPM7. We show that removing extracellular divalent cations unmasks large inward and outward monovalent currents, which can be inhibited by intracellular Mg2+. Inward currents are suppressed upon replacing extracellular Na+ by NMDG+. Divalent cations block monovalent IMIC and, at 10-20 mm, carry measurable currents. Their efficacy sequence in decreasing outward IMIC (Ni2+ = Mg2+ > Ca2+ > Ba2+) and in inducing inward IMIC (Ni2+ >> Mg2+ = Ca2+ approximately Ba2+), and their permeabilities calculated from reversal potentials are similar to those of TRPM6 and TRPM7 channels. The trivalent cations Gd3+ and Dy3+ also block IMIC in a voltage-dependent manner (delta = 0.4-0.5). In addition they inhibit the inward current carried by divalent cations. IMIC is regulated by pH. Decreasing or increasing extracellular pH decreased and increased IMIC, respectively (pH0.5 = 6.9, nH = 0.98). Qualitatively similar results were obtained on IMIC in rat basophilic leukaemia cells. These effects in cardiac myocytes were absent in the presence of high intracellular buffering by 40 mm Hepes. Our results suggest that IMIC in cardiac cells is due to TRPM channels, most probably to TRPM6 or TRPM7 channels or to their heteromultimeres.