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(Circulation Research. 2003;92:888.)
© 2003 American Heart Association, Inc.

Cellular Biology

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

Michael Poteser, Ichiro Wakabayashi, Christian Rosker, Margot Teubl, Rainer Schindl, Nikolai M. Soldatov, Christoph Romanin, Klaus Groschner

From the Department of Pharmacology and Toxicology (M.P., C. Rosker, M.T., K.G.), Karl-Franzens-University Graz, Austria; Department of Hygiene and Preventive Medicine (I.W.), School of Medicine, Yamagata University, Yamagata, Japan; Department of Biophysics (R.S., C. Romanin), Johannes-Kepler-University Linz, Austria; and the National Institute of Aging (N.M.S.), National Institutes of Health, Baltimore, Md.

Correspondence to Dr Klaus Groschner, Dept of Pharmacology and Toxicology, Karl-Franzens-University Graz, A-8010 Graz, Austria. E-mail klaus.groschner{at}uni-graz.at

	Abstract

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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⁺ (P_Ca/P_Na 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.

Key Words: intracellular pH • Ca²⁺ channels • nonselective cation channels • 2-APB • smooth muscle

	Introduction

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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 family^11–13 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

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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-CaM_Q, a fusion protein composed of an inactive CaM derivative and the green fluorescent protein derivative YFP. In CaM_Q, 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⁺ (Ca_i), and in some experiments, absolute values of Ca_i were determined according to Grynkiewicz et al¹⁸ as described previously.¹⁹ Intracellular pH levels (pH_i) were determined by measurement of pH-sensitive BCECF fluorescence ratios as described.¹⁹

Electrophysiology
The extracellular solutions for different experimental conditions were composed as given in the Table.

View this table: [in this window] [in a new window]   Table 1. Extracellular Bath Solutions

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 M 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 P_Ca/P_Na was calculated from reversal potential shifts obtained by replacement of external Na⁺ by Ca²⁺ according to the following equation: equation

Statistical Analysis
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.

	Results

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NH₄Cl-Induced Elevation of Intracellular pH and Ca²⁺ in A7r5 Smooth Muscle Cells
Figure 1A illustrates a typical time course of intracellular pH (pH_i) 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 pH_i and is not a simple consequence of Ca²⁺ release.

View larger version (19K): [in this window] [in a new window]   Figure 1. NH4Cl increases intracellular pH and cytoplasmic Ca2+ in A7r5 cells. Time courses of pHi and intracellular free Ca2+ recorded in BCECF-(A) and fura-2–loaded A7r5 cells (B). Extracellular administration of 20 mmol/L NH4Cl is indicated. Mean±SEM derived from 21 and 18 cells are shown.

View larger version (26K): [in this window] [in a new window]   Figure 2. NH4Cl mobilizes intracellular Ca2+ and promotes a Ca2+ entry pathway. Time courses of NH4Cl-induced changes in intracellular free Ca2+ of fura-2–loaded A7r5 cells. A, Cells were challenged with 20 mmol/L NH4Cl in Ca2+ containing bath solution, in the absence of Ca2+ channel blockers (control) and in the presence of 10 µmol/L verapamil or 200 µmol/L 2-APB. B and C, Cells were challenged with 20 mmol/L NH4Cl in Ca2+-free solution, and extracellular Ca2+ was elevated to 1 mmol/L (+Ca2+) as indicated. Experiments were performed with nonstimulated cells (control) and in cells exposed to NH4Cl for the indicated time. B, Effect of short-term exposure to NH4Cl; C, Effect of continuous exposure to NH4Cl throughout the Ca2+ readdition protocol. Extracellular administration of 20 mmol/L NH4Cl is indicated. Mean±SEM derived from 20 to 35 cells are given.

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).

View larger version (22K): [in this window] [in a new window]   Figure 3. NH4Cl activates a voltage-independent, Ca2+-permeable conductance in A7r5 cells. A, Time course of NH4Cl-induced inward current at -70 mV recorded by the perforated-patch technique. B, Current responses to individual voltage-ramp depolarization before (1) and after (2) administration of 20 mmol/L NH4Cl corresponding to A. Insert shows the net NH4Cl-induced current derived by subtraction (2-1). Extracellular solution was Na+-free (0 Na+/2 Ca2+) and contained 10 µmol/L verapamil; the pipette solution contained K+ as the main charge carrier. C, Current-to-voltage relation of NH4Cl-induced currents derived by subtraction of current responses obtained by voltage-ramp stimulation in the presence and absence of NH4Cl. Experiments were performed with Cs+ in the pipette and with the indicated bath solutions. D, Zero current potentials measured in extracellular solutions of different Na+ and Ca2+ concentrations as indicated. Bath solutions were supplemented with 10 µmol/L verapamil. Asterisks indicate statistically significant differences. Mean±SEM are given (n=4).

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 P_Ca/P_Na 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.

View larger version (12K): [in this window] [in a new window]   Figure 4. NH4Cl-induced activation of the voltage-independent cation conductance is associated with inhibition of L-type Ca2+ currents. A, Current-to-voltage relationship derived from a sequence of voltage steps before (1) and after (2) administration of 20 mmol/L NH4Cl in physiological bath solution (137 Na+/2 Ca2+). B, Individual current traces before and after administration of NH4Cl obtained with voltage-steps from -70 to +10 mV. Experiments were performed using K+ pipette solution.

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 currents^4,8 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 (CaM_Q), 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 CaM_Q (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 CaM_Q further substantiates a pivotal role of Ca²⁺ in the effects of intracellular alkalosis on membrane conductances of A7r5 cells.

View larger version (21K): [in this window] [in a new window]   Figure 5. NH4Cl-induced inhibition of L-type currents is dependent on extracellular Ca2+ and mediated by a Ca2+/CaM-mediated mechanism. NH4Cl effects on membrane conductance recorded with 10 Ba 2+/0.02 Ca2+ (A) and with 10 Ba2+/2 Ca2+ (B) bath solution. C, Effect of NH4Cl in a cell expressing CaMQ using 137 Na+/2 Ca2+ bath solution. Top panels, Current responses to voltage-ramp depolarizations before and during perfusion with 20 mmol/L NH4Cl (+ NH4Cl). Bottom panels, Time course of NH4Cl-induced changes in L-type Ba2+ current measured at a test potential of 0 mV. Experiments were performed with Cs+ in the pipette solution.

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.

View larger version (26K): [in this window] [in a new window]   Figure 6. 2-APB inhibits NH4Cl-induced voltage-independent cation conduction and reduction of L-type Ca2+ currents without direct modulation of L-type channels. A, Current responses to individual voltage-ramp depolarization before and after administration of 20 mmol/L NH4Cl (+NH4Cl) in the absence (left) and presence of 200 µmol/L 2-APB (right). 137 Na+/2 Ca2+ was used in the bath solution and Cs+ was the main charge carrier in the pipette. B, Mean values of NH4Cl-induced increases in the inward currents at -70 mV (left) and concomitant reduction of peak L-type currents (right) in the absence (control) and presence of 200 µmol/L 2-APB. Asterisks indicate statistically significant differences. Mean±SEM are given (n=5 to 7). C, Current responses to individual voltage-ramp depolarization before and after administration of 20 mmol/L NH4Cl (+NH4Cl) in presence of 200 µmol/L 2-APB and 10 Ba2+/0 Ca2+ bath solution (+2-APB/Ba2+).

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).

View larger version (18K): [in this window] [in a new window]   Figure 7. 2-APB reduces AVP-induced voltage-independent cation conduction and reduction of L-type Ca2+-currents. A, Current responses to individual voltage-ramp depolarization before and after administration of 10 µmol/L AVP (+ AVP) in the absence (left) and presence of 200 µmol/L 2-APB (right). 137 Na+/2 Ca2+ was used as bath solution, and Cs+ was the main charge carrier in the pipette. B, AVP-induced increases in the inward currents at -70 mV (left) as well as concomitant reduction of peak L-type currents (right) in the absence (control) and presence of 200 µmol/L 2-APB. Asterisks indicate statistically significant differences. Mean±SEM are given (n=5).

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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 pH_i-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 (CaM_Q) 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 CaM_Q, 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.

	Acknowledgments

 
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 CaM_Q cDNA, respectively.

Received August 19, 2002; revision received February 6, 2003; accepted March 18, 2003.

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