Crosstalk Between Voltage-Independent Ca2+ Channels and L-Type Ca2+ Channels in A7r5 Vascular Smooth Muscle Cells at Elevated Intracellular pH
Evidence for Functional Coupling Between L-Type Ca2+ Channels and a 2-APB–Sensitive Cation Channel
This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 μmol/L) but not by verapamil (10 μmol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa ≈6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C–coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.
The general impact of intracellular pH on smooth muscle Ca2+ homeostasis is well established,1–3 and the principle sensitivity of key Ca2+ entry pathways such as L-type Ca2+ channels and store-operated Ca2+ channels to regulation by intracellular pH has been demonstrated.4–6 In contrast to voltage-dependent L-type channels, which have extensively been studied in terms of sensitivity to cytosolic pH,4,7,8 little is known about the effects of intracellular pH on voltage-independent channels, which are likely to contribute to stimulated Ca2+ entry in vascular smooth muscle.9–11 The observed effects of alkalinization on intracellular Ca2+ stores suggest pH-sensitivity of store-operated Ca2+ entry. Such pathways may be provided by cation channels of the classical TRP family11–13 or of other, as yet unidentified, store-operated channel proteins.
In the present study, we investigated the effects of elevation of intracellular pH on membrane conductances of A7r5 vascular smooth muscle cells. This cell line expresses at least three types of Ca2+ entry pathways that are typical for vascular smooth muscle and represent potential targets for modulation by intracellular pH: voltage-gated Ca2+ channels,14 store-operated Ca2+ entry channels,15 and nonselective, Ca2+-permeable cation channels.9
We report for the first time on a crosstalk between voltage-gated L-type Ca2+ channels and nonselective Ca2+-permeable cation channels of vascular smooth muscle and suggest a tight functional interaction between these cation channels.
Materials and Methods
Cell Culture, cDNA Constructs, and Cell Transfection
Rat aortic smooth muscle cells (A7r5) were cultured at 37°C in DMEM (Dulbecco’s Modified Eagle’s Medium) containing 10% FCS (fetal calf serum) and 100 U/mL penicillin as well as 100 μg/mL streptomycin. Cells were used for experiments in a subconfluent state of growth. To express a dominant-negative mutant of calmodulin, A7r5 cells were transfected by electroporation to transiently express YFP-CaMQ, a fusion protein composed of an inactive CaM derivative and the green fluorescent protein derivative YFP. In CaMQ, all four EF-hand motifs are mutated.16,17 The N-terminally tagged YFP-CaM(Q) was prepared by 3-piece ligation. For this, the 720-bp BsrGI (blunt)/KpnI fragment of the pEYFP vector (Clontech), and the 450-bp SalI (blunt)/BglII fragment of CaM(Q)-pBF17 were ligated into the pcDNA3 vector at KpnI and BamHI sites. The entire coding sequence has been verified by DNA sequencing. Cells transfected by the same procedure to express YFP only were used for control experiments.
HEK293 cells stably transfected to express the murine TRPC6 protein (Q61143). Cells were cultured in DMEM supplemented with 0.2 g/mL Geneticin. Control experiments were performed in HEK293 cells stably transfected with the neomycin resistance cassette only.
Measurement of Intracellular Calcium and pH in Single Cells
A7r5 cells were loaded with fura-2-AM (Molecular Probes) for cytosolic calcium estimation or with tetra-acetoxymethyl ester of 2,7-bis-carboxyethyl-5,6-carboxy fluorescin (BCECF-AM, Lambda Fluorescence Technology) for detection of pH changes. Cells were constantly perfused during experiments with buffer containing (in mmol/L) 137 NaCl, 5,4 KCl, 15 HEPES, and ±2 CaCl2. Excitation light was supplied via a Polychrome II polychromator (TILL Photonics), and emission was detected by a Sensicam CCD-camera (PCO Computer Optics). Fura-2-AM–induced cell fluorescence was measured ratiometrically at 340 and 380 nm excitation wavelengths and emission collected at 510 nm. BCECF-AM fluorescence excitation wavelengths were 500 nm and 415 nm and emission detected at 530 nm. Digital image recordings were evaluated using Axon Imaging Workbench (Axon Instruments). Changes in Ca2+-sensitive fluorescence ratio were used to analyze and compare changes in intracellular Ca2+ (Cai), and in some experiments, absolute values of Cai were determined according to Grynkiewicz et al18 as described previously.19 Intracellular pH levels (pHi) were determined by measurement of pH-sensitive BCECF fluorescence ratios as described.19
The extracellular solutions for different experimental conditions were composed as given in the Table.
The pipette solution contained (in mmol/L) 120 Cs-methanesulfonate, 20 CsCl, 15 HEPES, 3 EGTA with pH of 7.4 adjusted with N-methylglucamine (NMDG) designated as Cs+ pipette solution. Some experiments were performed with a pipette solution containing (in mmol/L) 110 K-gluconate, 10 KCl, 5 MgCl2, 10 HEPES, and 10 EGTA with pH of 7.4 adjusted with NMDG, designated as K+ pipette solution. Experiments were performed with the perforated-patch technique using amphotericin B (Sigma; dissolved in DMSO (dimethylsulfoxide) at a final concentration of 40 to 80 μg/mL) in the pipette solution.
Pipettes (3 to 3.5 MΩ were pulled from borosilicate glass (Clark Electromedical Instruments). Currents were recorded at room temperature using an EPC-7 patch clamp amplifier (List). Voltage-clamp protocols (voltage ramps from −100 to +80 mV/0.6 V/s, 0.2 Hz, holding potential −70 mV) were controlled by pClamp software using a Digidata 1200 Computer Interface (Axon Instruments). The permeability ratio PCa/PNa was calculated from reversal potential shifts obtained by replacement of external Na+ by Ca2+ according to the following equation: equation
Averaged data are illustrated as mean±SEM obtained from the indicated number of experiments. Differences were considered statistically significant at P<0.05 using Student’s t test for unpaired values.
NH4Cl-Induced Elevation of Intracellular pH and Ca2+ in A7r5 Smooth Muscle Cells
Figure 1A illustrates a typical time course of intracellular pH (pHi) measured in BCECF-loaded A7r5 cells during extracellular administration and removal of NH4Cl. NH4Cl (20 mmol/L) induced a rapid increase in intracellular pH from about 7.2 to 7.6, which remained fairly stable in the presence of NH4Cl and declined rapidly below baseline levels on NH4Cl removal, reflecting some NH4Cl wash-out acidification. Figure 1B shows typical changes in intracellular Ca2+ associated with NH4Cl-induced alkalosis in the presence of extracellular Ca2+. NH4Cl-administration resulted in a rapid elevation of intracellular Ca2+ displaying a transient peak of about 600 nmol/L followed by a slowly declining plateau phase as illustrated in Figure 1B. Intracellular Ca2+ declined to resting levels on removal of NH4Cl. Verapamil, a classical blocker of voltage-gated L-type channels, failed to affect the NH4Cl-induced Ca2+ signals significantly as shown in Figure 2A. By contrast, 2-APB, an inhibitor of store-operated Ca2+ entry channels and of IP3 receptors, suppressed the NH4Cl-induced sustained elevation of intracellular Ca2+ in a concentration-dependent manner (10 to 200 μmol/L). At 200 μmol/L, 2-APB abolished the plateau phase while an initial transient elevation of Ca2+ remained (Figure 2A). 2-APB (200 μmol/L) did not affect NH4Cl-induced modulation of pHi (n=4; not shown). The effects of high NH4Cl concentrations were not due to changes in osmolarity because equal osmotic changes introduced with TEACl were without effect on intracellular Ca2+ of A7r5 cells, and significant 2-APB–sensitive Ca2+ entry was observed with NH4Cl concentrations as low as 5 mmol/L (n=3; not shown). Transient elevation of intracellular Ca2+ by NH4Cl persisted in the absence of extracellular Ca2+, whereas sustained elevation of intracellular Ca2+ required the presence of extracellular Ca2+ (Figures 2A and 2B). To test for pH-dependent activation of a store-operated Ca2+ entry pathway, we performed classical Ca2+ readdition protocols, as illustrated in Figure 2B. Ca2+ mobilization by NH4Cl itself was not sufficient to activate the Ca2+ entry pathway, as Ca2+ readdition did not result in enhanced Ca2+ entry when NH4Cl was removed after discharge of Ca2+ stores (Figure 2B). Figure 2C illustrates stimulation of Ca2+ entry in the continuous presence of NH4Cl. Thus, activation of the 2-ABP–sensitive Ca2+ entry pathway requires persistent elevation of pHi and is not a simple consequence of Ca2+ release.
Electrophysiological Characterization of the NH4Cl-Induced Ca2+ Entry Pathway
To characterize the membrane conductance underlying the NH4Cl-induced Ca2+ entry pathway, we performed perforated-patch, whole-cell clamp experiments initially using a Ca2+ containing extracellular solution supplemented with 1 μmol/L verapamil to block voltage-dependent L-type channels. A typical time course of currents recorded with Ca2+ as the main extracellular cation at a membrane potential of −70 mV as well as representative current to voltage relations are depicted in Figures 3A and 3B. NH4Cl induced an increase in inward currents at negative membrane potential that reversed rapidly on washout of NH4Cl. NH4Cl-induced membrane currents reversed at about +10 mV with K+ as the main intracellular cation in the pipette solutions (Figure 3B). Similar reversal potentials were observed when Cs+ replaced K+ in the pipette solution. Figure 3C compares the net NH4Cl-induced membrane currents recorded in the presence of 1 μmol/L verapamil with approximately 20 μmol/L and 2 mmol/L extracellular Ca2+ and Cs+ as the main cation in the pipette solution. In these experiments, extracellular Na+ was replaced by TEA (0 Na+/0.02 Ca2+ and 0 Na+/2 Ca2+). Reduction of extracellular Ca2+ to ≈0.02 mmol/L resulted in a substantial suppression of NH4Cl-induced currents and the residual current reversed close to the calculated equilibrium potential for Cs+ (−68 mV). These results demonstrate that the NH4Cl-activated Ca2+ entry pathway is based on a Ca2+ permeable nonselective cation channel. The reduced outward currents suggests some additional Ca2+ dependence of channel gating. The observed Ca2+ conductance did not display significant voltage dependence. To characterize the current-to-voltage relation of this Ca2+ conductance in the absence of contaminating voltage-gated Ca2+ currents and without the use of a pharmacological tool, we performed experiments using reversed voltage ramps and a holding potential of 0 mV to inactivate T- and L-type Ca2+ channels. The current-to-voltage relation obtained with the reversed voltage ramp protocol was consistent with that observed with depolarizing voltage ramps in the presence of verapamil (n=2; not shown).
The nonselective nature of the NH4Cl-activated conductance was clearly evident when the currents were recorded with extracellular Na+ as the main charge carrier and with Cs+ in the pipette solution. Under these conditions, NH4Cl-induced currents reversed at +50±6 mV (n=5) in 137 mmol/L extracellular Na+ and at +12±5 mV (n=4) in 10 mmol/L extracellular Na+. Figure 3D summarizes the reversal potentials of the NH4Cl-induced nonvoltage-dependent current component measured in different extracellular solutions. Based on the reversal potentials measured with 2 mmol/L Ca2+ (+5 mV) and with 137 mmol/L Na+ (+50 mV), a PCa/PNa permeability ratio of 6 was calculated.
NH4Cl-Induced Ca2+ Entry Is Associated With Suppression of L-Type Ca2+ Currents
In the absence of the verapamil and in most physiological conditions, with minimized dialysis of the cells achieved by the perforated-patch technique, NH4Cl (20 mmol/L) induced not only inward currents at negative potentials but also a marked reduction in the inward currents through L-type Ca2+ channels recorded at potentials positive to −20 mV (Figure 4). The current to voltage relation obtained from a sequence of voltage steps recorded in the absence and presence of 20 mmol/L NH4Cl is shown in Figure 4A. The concomitant increase in inward currents at negative potentials and the decrease in inward currents at positive potentials is evident from both the current to voltage relation shown in Figure 4A and the current traces derived from depolarizing voltage steps shown in Figure 4B. NH4Cl increased the holding current recorded at −70 mV and suppressed the typical voltage gated L-type current without changing the current to voltage relation essentially. The depolarization required for maximum L-type inward currents remained unaffected (about +10 mV) during exposure of cells to NH4Cl. Figure 4B illustrates the effects of NH4Cl on membrane currents measured at the holding potential of −70 mV and during depolarizing voltage steps. Concomitant changes in holding current and voltage-gated inward current are evident.
Effects of NH4Cl on Voltage-Gated L-Type Ca2+ Channels Conductances of A7r5 Smooth Muscle Cells Are Mediated by Ca2+
NH4Cl-induced alkalinization of smooth muscle cells has been reported to potentiate L-type channel currents4,8 in experimental conditions that minimize Ca2+-mediated feedback regulation of the channels. To analyze this discrepancy, and to test for a possible role of a Ca2+ entry-mediated negative feedback in the observed co-regulation of voltage-independent and voltage-dependent Ca2+ channels, we performed two sets of experiments: (1) experiments in which extracellular Ca2+ was replaced as a charge carrier by Ba2+, and (2) experiments with Ca2+ as charge carrier and cells expressing a dominant-negative mutant of calmodulin (CaMQ), which is known to prevent Ca2+/CaM-induced inactivation of L-type currents.16 Using Ba2+ (10 mmol/L) as the main charge carrier at about 0.02 mmol/L Ca2+ (nominally Ca2+-free), we observed a marked NH4Cl-induced increase in L-type current as illustrated in Figure 5A. A similar result was obtained when extracellular Ca2+ was chelated to allow Na+ permeation20 through the L-type channel (n=3; not shown). Addition of Ca2+ (2 mmol/L) to the 10 mmol/L Ba2+ recording solution recovered inhibition of L-type currents by NH4Cl (Figure 5B), demonstrating a tight link between Ca2+ entry and inhibition of L-Type currents. Importantly, NH4Cl failed to inhibit but clearly potentiated L-type Ca2+ currents measured with 2 mmol/L extracellular Ca2+ in cells expressing CaMQ (Figure 5C; n=6), but not in sham-transfected controls (n=3; not shown), which displayed responses similar to nontransfected cells. The sensitivity of NH4Cl regulation of L-type channels to CaMQ further substantiates a pivotal role of Ca2+ in the effects of intracellular alkalosis on membrane conductances of A7r5 cells.
Ca2+ Entry Through 2-APB–Sensitive Voltage-Independent Cation Channels Is a Key Determinant of L-Type Ca2+ Channel Function in A7r5 Smooth Muscle Cells
The described experimental results led us to hypothesize that the NH4Cl-induced inhibitory regulation is mediated by Ca2+ entry through the alkalosis-activated nonselective cation channel. To further test this hypothesis, we aimed at blocking the nonselective cation conductance to reveal its possible relevance for inhibition of L-type currents. 2-APB (200 μmol/L), which suppressed NH4Cl-induced Ca2+ entry in Fura-2 experiments (see Figure 2), inhibited the nonselective cation conductance and abolished the sensitivity of L-type currents to inhibition by NH4Cl (Figures 6A and 6B), suggesting a tight coupling between these two conductances. Figure 6C shows a summary of the effects of 2-APB on inward currents at −70 mV and on peak L-type currents. 2-APB by itself neither inhibited L-type currents nor affected the potentiation of Ba2+ currents through L-type channels (Figure 6C). Thus, 2-APB selectively suppressed a mechanism of Ca2+-dependent control of smooth muscle L-type currents.
Inhibitory regulation of voltage-gated L-type channels has also been reported for stimulation of phospholipase C–coupled receptors, which is typically associated with activation of nonselective cation conductances in A7r5 cells.21 To test the hypothesis that the observed functional coupling between 2-APB–sensitive cation channels and L-type channels is a more general phenomenon that is not limited to intracellular alkalosis, we performed experiments with arginine8-vasopressin (AVP). Similar to NH4Cl, AVP (1 μmol/L) induced concomitant activation of a nonselective conductance and suppression of L-type currents (Figure 7A).
2-APB suppressed the AVP-induced nonselective conductance and eliminated the inhibitory regulation of L-type currents as shown in Figure 7B. Thus, both intracellular alkalosis and AVP activate a 2-APB–sensitive conductance that effectively controls voltage-gated Ca2+ channels. AVP (1 μmol/L) failed to increase inward membrane currents in the presence of NH4Cl (20 mmol/L; n=6, not shown; provided in the online data supplement available at http://www.circresaha. org). Neither AVP nor NH4Cl was able to produce significant further (additive) current increases after preactivation of nonselective channels by the other agent, indicating that the same type of channel may be activated by AVP and NH4Cl. Because TRPC6 was recently proposed to form at least part of the AVP-activated smooth muscle Ca2+ entry pathway, we tested the sensitivity of heterologously expressed TRPC6 channels. We observed that HEK293 cells overexpressing TRPC6, in contrast to vector-transfected controls, displayed a small transient NH4Cl-induced cation conductance, which exhibited inward rectification (n=6, not shown; see online data supplement). Thus, the current to voltage relation of the TRPC6 generated conductance was different from that of the nonselective conductance observed in A7r5.
Our results suggest that the pH-sensitive, nonselective cation channel of A7r5 cells is permeable for Ca2+ and controls local intracellular Ca2+ gradients that determines L-type Ca2+ channel function.
Our findings demonstrate that, intracellular alkalinization causes activation of a 2-APB–sensitive, Ca2+-permeable cation channel in A7r5 cells, and that activation of this channel is associated with suppression of L-type Ca2+ channels. We suggest coregulation of these two distinct Ca2+ entry pathways in terms of a crosstalk that serves fine-tuning of smooth muscle Ca2+ signaling.
Intracellular Alkalinization Activates a Nonselective Ca2+-Permeable Cation Conductance in A7r5 Smooth Muscle Cells
Ca2+ permeability of the alkalosis-induced membrane conductance was clearly evident from an approximately 80-mV negative shift in reversal potentials associated with a reduction of extracellular Ca2+ by two orders of magnitude (0 Na+/2 Ca2+ versus 0 Na+/0.02 Ca2+ solution). The cation channels underlying this conductance were barely voltage-dependent and insensitive to the classical Ca2+ channel blocker verapamil but sensitive to inhibition by 2-APB, which is known to interfere with IP3 receptor function22 and to inhibit store-dependent Ca2+ entry pathways.23 NH4Cl-induced, 2-APB–sensitive Ca2+ entry was also observed in primary rat aortic cells (online data supplement), and is therefore unlikely a phenomenon specific for the A7r5 cell line. Effects of 2-APB may be mediated by a direct block of the Ca2+ entry channels as recently suggested for capacitative Ca2+ entry.23 Interestingly, NH4Cl-induced intracellular Ca2+ mobilization in Ca2+ free solution was not significantly inhibited by 2-APB (200 μmol/L), arguing against a role of IP3 in NH4Cl-induced Ca2+ mobilization. Indeed, we observed that NH4Cl is able to mobilize Ca2+ even after depletion of IP3-sensitive stores with AVP (1 μmol/L) or the SERCA inhibitor thapsigargin (100 nmol/L) as well as in the presence of 1 μmol/L ryanodine (unpublished data, 2003). The existence of nonmitochondrial, IP3, and/or thapsigargin insensitive intracellular Ca2+ pools has recently been suggested,24,25 and the nature of these pools in A7r5 cells has so far not been unraveled. Thus, at present the intracellular Ca2+ pool targeted by NH4Cl remains elusive. Nonetheless, our results clearly demonstrate that sustained intracellular alkalosis is essential for activation of the NH4Cl-induced Ca2+ entry, whereas depletion of intracellular Ca2+ stores by NH4Cl is not sufficient to trigger Ca2+ entry and the associated modulation of L-type Ca2+ channels.
A Ca2+/Na+ permeability ratio of ≈6 was determined for the NH4Cl-induced cation conductance, ie, a value similar to that reported for TRPC6 channels,26 which have been suggested to play a role in the vasopressin-induced cation conductances of A7r5.27 However, the alkalosis-activated Ca2+ channels displayed some properties different from those reported for TRPC6 channels such as insensitivity to stimulation by flufenamate and relatively poor Ba2+ permeability as indicated from Fura-2 experiments (unpublished observations, 2003). Moreover, our experiments with HEK293 cells overexpressing TRPC6 revealed that expression of this protein itself is not sufficient to generate a pH-sensitive cation resembling that of A7r5 cells. Thus, the described alkalosis-regulated cation channel is unlikely identical with the TRPC6 cation channels characterized in heterologous expression systems. Nonetheless, we cannot exclude the involvement of TRPC6 or a related TRP protein in the pH-regulated Ca2+ conductance of A7r5 cells, in terms of a component of heteromultimeric pH-sensitive cation channels.
It remains to be clarified whether pHi-dependent activation of these cation channels involves a direct deprotonation of channel proteins or associated regulatory proteins. At present, we cannot exclude that elevation of pH triggers a more complex cascade of events leading to activation of voltage-independent Ca2+-permeable channels
Intracellular Alkalinization Inhibits L-Type Channels in A7r5 Smooth Muscle Cells
Previous studies have demonstrated that elevation of intracellular pH represents a potential stimulatory factor for L-type Ca2+ channels.4 Promotion of L-type Ca2+ channel activity by intracellular alkalosis has previously been observed with Ba2+ as charge carrier in experiments using a high intracellular Ca2+ buffer.4 In the present study, we demonstrate that the alkalosis-induced stimulation of L-type Ca2+ channels occurs when either Ca2+ entry is minimized by use of Ba2+ or Na+ as charge carrier, or when Ca2+/CaM-mediated negative feedback regulation is suppressed. Addition of 2 mmol/L Ca2+ to an extracellular solution containing 10 mmol/L Ba2+ extracellular solution was sufficient to enable NH4Cl-induced inhibition of L-type channels. It appears reasonable to assume that under these conditions, significant Ca2+ entry takes place via Ca2+ permeation through the voltage-independent channels. Consequently, it is tempting to speculate that the inhibitory effects of NH4Cl are mediated by Ca2+ entry and the built-up of intracellular Ca2+ gradients, leading to classical Ca2+-induced feedback inhibition of L-type channels.28,29 This concept was confirmed by experiments with cells, which were transfected to express a dominant-negative mutant of calmodulin (CaMQ) in order to eliminate Ca2+/CaM-induced feedback inhibition of L-type channels in intact A7r5 cells. NH4Cl failed to inhibit L-type channels in cells that expressed CaMQ, as expected for a mechanism involving Ca2+/CaM-dependent negative feedback regulation. Our present data indicate the existence of a mechanism that efficiently counteracts the promotion of voltage-dependent Ca2+ entry due to membrane depolarization and direct deprotonation of Ca2+ channel proteins during intracellular alkalosis. This mechanism may be of importance to prevent excessive Ca2+ loading and enables fine-tuning of Ca2+ entry.
2-APB Reveals Crosstalk Between Voltage-Independent and Voltage-Dependent Ca2+ Entry Channels
2-APB (at 200 μmol/L) did not affect L-type channels directly but inhibited NH4Cl-induced voltage-independent currents and blunted the inhibitory modulation of L-type channels by NH4Cl. Our results suggest a novel concept of Ca2+ entry control via tight interaction between voltage-independent and voltage-dependent Ca2+ channels. These different Ca2+ entry channels appear to communicate via local Ca2+ signaling events and may thus be located in proximity.
In A7r5 cells, an AVP-induced voltage-independent cation conductance is typically associated with persistent suppression of L-type currents. So far, these two AVP-induced parallel changes in membrane conductances were considered as rather independent cellular events, with inhibition of L-type current resulting mainly from protein kinase C–mediated inhibition of L-type channels. Our finding that 2-APB is a selective blocker of voltage-independent Ca2+ conductances in A7r5, without direct effects on L-type channels, prompted us to evaluate the extent of functional coupling between the AVP-induced voltage-independent Ca2+ entry and L-type channels. 2-APB completely abolished suppression of L-type currents during stimulation of A7r5 cells with AVP. These results are consistent with the concept that Ca2+ entry through nonselective, 2-APB–sensitive channels constitutes a mechanism that is essential for regulation of L-type channels. It is tempting to speculate that AVP and NH4Cl activate the same channel protein or two tightly related channels. This hypothesis is supported by the lack of additive conductance increase by either agent in the presence of the other agent. However, modulation of membrane conductances during enhanced phospholipase C activity is difficult to interpret. Multiple signaling cascades are turned on during stimulation of phospholipase C–coupled receptors, and it has been convincingly demonstrated that AVP-induced voltage-independent cation conductances and Ca2+ entry involve several components most likely including a classical store-operated Ca2+ entry pathway.9,10,30 It appears reasonable to conclude that a phospholipase C–dependent Ca2+ entry pathway that is sensitive to inhibition by 2-APB is tightly linked to voltage-gated L-type channels. It remains to be clarified whether this phospholipase C–dependent Ca2+ entry pathway and the alkalosis-activated Ca2+ entry involve the same channel protein.
In aggregate, this study provides evidence for the existence of a voltage-independent 2-APB–sensitive Ca2+ channel, which is activated during elevation of intracellular pH. Ca2+ entry through these channels is associated with inhibition of L-type channels. We suggest the control of L-type channels by Ca2+ entry through voltage-independent cation channels as an important protective mechanism to avoid cytotoxic Ca2+ loading and as an attractive target for drug therapy.
This study was supported by project FWF-F715 (SFB Biomembranes), FWF-14950, and ÖNB-8216 to K.G. and FWF15387 and ÖNB 9343 to C. Romanin. The authors with to thank Drs. M.X. Zhu, J.P. Adelman, and J. Maylie for providing TRPC6 and CaMQ cDNA, respectively.
- Received August 19, 2002.
- Revision received February 6, 2003.
- Accepted March 18, 2003.
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