Introduction

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Voltage-gated Ca²⁺ channels are a major route for Ca²⁺ entry into cells in response to stimulation by hormones, neurotransmitters, or drugs. The resulting rise in cytoplasmic free Ca²⁺ triggers a cascade of intracellular signaling events, which underlie a variety of cellular responses, ranging from contraction and secretion to growth and mitogenesis. Therefore, identification of the molecular basis for functional coupling between Ca²⁺ channels and hormone or neurotransmitter receptors may provide critical information on cellular signaling mechanisms.

The cardiac L-type Ca²⁺ channel is composed of the pore-forming _1C and auxiliary  and ₂/ subunits (Catterall, 1995). In an artificial expression system, the _1C2/ complex is sufficient to give rise to Ca²⁺ channels exhibiting all of the major electrophysiological properties observed in vivo. However, functional regulation of the recombinant Ca²⁺ channel remains largely unknown. For example, in cardiac or vascular cells, the _1C channel is modulated by protein kinase A- and protein kinase C-dependent phosphorylation (McDonald et al., 1994). However, when all three recombinant subunits of the channel are coexpressed in Xenopus laevis oocytes or in eukaryotic systems (Chinese hamster ovary or human embryonic kidney cells), their modulation through phosphorylation is either strongly reduced or essentially absent (Bouron et al., 1995; Zong et al., 1995; Shuba et al., 1997), even though the expressed channels display the same voltage dependence, gating kinetics, unitary conductance, and pharmacological properties as the native _1C L-type Ca²⁺ channels. These findings demonstrate the complexity of molecular signaling involving the _1C Ca²⁺ channels; this complexity extends to the largely unexplored area of "cross-talk" between recombinant _1C channels and hormone receptors that are coexpressed in X. laevis oocytes.

The coupling of _1C Ca²⁺ channels with angiotensin II AT₁ receptors has attracted much attention. For example, the L-type Ca²⁺ channel blockers verapamil, diltiazem, and nifedipine have been shown to block angiotensin II-mediated vascular contraction in vivo in humans (Andrawis et al., 1992). Activation of AT₁ receptors seems to be associated with both immediate contractile and long term growth responses in vascular smooth muscle and cardiac myocytes (Baker et al., 1992; Sadoshima and Izumo, 1993; Miyata and Haneda, 1994). Supporting the possibility of interactions between the G protein-coupled AT₁ receptors (Anand-Srivastava, 1983; Ohya and Spereliakis, 1991) and voltage-activated Ca²⁺ channels is the regulation of neuronal (Scott and Dolphin, 1987) and cardiac (Yatani et al., 1987) L-type Ca²⁺ channels by PTX-sensitive or -insensitive G proteins. Similar interactions have been suggested for angiotensin II activation of L-type Ca²⁺ currents in rat portal vein myocytes (Macrez-Lepretre et al., 1996) and T-type Ca²⁺ currents in adrenal zona glomerulosa cells (Lu et al., 1996).

In this study, we have used the X. laevis oocyte expression system to study the functional coupling between recombinant rat AT_1A receptors and splice variants of recombinant human _1C Ca²⁺ channels with or without the molecular motif responsible for Ca²⁺-dependent inactivation of the channel. We show that heterogeneously expressed Ca²⁺ channels and AT_1A receptors are functionally coupled via the G protein/IP₃-mediated Ca²⁺ signaling cascade. Additionally, we report that the molecular locus for the angiotensin-induced modulation of the _1C Ca²⁺ channel is independent of permeation of Ca²⁺ through the pore and is confined to the carboxyl-terminal cytoplasmic motif (positions 1572-1651), which contains multiple Ca²⁺ sensors of the channel.

	Materials and Methods

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Preparation of mRNAs. Template _1C,77 (Soldatov et al., 1995) and _1C,86 (Soldatov et al., 1997) cDNAs were linearized by digestion with BamHI. Capped transcripts were synthesized in vitro with T7 RNA polymerase, using the mRNA cap kit (Stratagene, La Jolla, CA). mRNAs were dissolved in water (0.5 µg/µl). Rat angiotensin AT_1A receptor (Murphy et al., 1991) transcripts were kindly provided by Kathryn Sandberg (Georgetown University).

Oocyte preparation and injection. Mature female X. laevis frogs were purchased from Xenopus I (Ann Arbor, MI). Clusters of oocytes were defolliculated by shaking for 2 hr at room temperature in 25 ml of medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, pH 7.5 (adjusted with NaOH), and 0.2% collagenase A (Boehringer Mannheim, Indianapolis, IN). Oocytes were injected with 50-100 nl of _1C,77 or _1C,86 mRNA premixed with mRNAs coding for auxiliary ₁ (Ruth et al., 1989) and ₂ subunits (Singer et al., 1991) and the AT_1A receptor (in a 1:1:1:0.05 molar ratio). Injected oocytes were incubated at 18° in sterile Barth's medium supplemented with 10,000 units/liter penicillin, 10 mg/liter streptomycin, 50 mg/liter gentamicin, and 0.5 mM theophylline (all from Sigma Chemical Co., St. Louis, MO).

Electrophysiological measurements. Whole-cell ion currents were recorded at room temperature (20-22°) by a two-electrode, voltage-clamp method, as previously described (Soldatov et al., 1998). Current traces were elicited at 30-sec intervals by 1-sec (current-voltage relationships) or 250-msec test pulses to +20 mV, from a holding potential of 90 mV. The Ba²⁺ extracellular (bath) solution contained 50 mM NaOH, 1 mM KOH, 10 mM HEPES, and 40 mM Ba(OH)₂ (pH adjusted to 7.4 with methanesulfonic acid). Voltage-clamped oocytes were continuously perfused with control experimental solutions at the rate of ~10 ml/min (bath volume, ~150 µl). Human angiotensin II (Sigma) was applied extracellularly. In some experiments, oocytes were injected with 50 nl of PTX (5 µg/ml), 10 mM GDPS, 94 mM Cs₄BAPTA (pH 7.4), or 10 µM heparin (molecular weight, ~3000; Sigma) approximately 1 hr before the experiment. In other experiments, oocytes were incubated at 18° overnight in Ca²⁺-free Barth's solution containing 10 nM thapsigargin (RBI, Natick, MA), to deplete their intracellular Ca²⁺ stores. Results are shown as mean ± standard error. I_Ba, determined in the presence of 5 µM (±)-PN200-110 to block the L-type current, did not exceed 3-5% of the total current.

	Results

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Coexpression of the _1C,77 channel with the AT_1A receptor allows regulation of Ca²⁺ channels by angiotensin. Coinjection into X. laevis oocytes of cRNAs coding for the conventional _1C,77 channel and auxiliary ₁ and ₂ subunits gave rise to the expression of well defined, slowly inactivating currents through Ca²⁺ channels 2-3 days after the injection of cRNAs (Soldatov et al., 1995). With Ba²⁺ as a charge carrier, step depolarization to +20 mV from a holding potential of 90 mV activated a slowly inactivating, L-type I_Ba (mean amplitude, 1.64 ± 0.33 µA, n = 9). Application of 0.5-10 µM angiotensin to oocytes expressing only _1C,77 Ca²⁺ channels produced little or no change in the magnitude or the kinetics of the current, at all voltages examined (Fig. 1, A and C; Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of angiotensin on Ca2+ currents. A, Representative traces of IBa through 1C,77 channels, elicited by stepwise depolarization to +20 mV from a holding potential of 90 mV, before () and 4 min after () application of 1 µM angiotensin. B, Traces of IBa through 1C,77 channels coexpressed with AT1A receptors, recorded before () and 1.5, 2, and 3 min after application of 1 µM angiotensin. C, Time dependence and reversibility of the angiotensin effect on IBa through 1C,77 channels expressed alone () or coexpressed with AT1A receptors (). IBa amplitudes were measured in response to 250-msec test pulses to +20 mV, applied at 30-sec intervals, and were normalized to maximal IBa in the absence of angiotensin. Arrows, times of application of bath solutions containing the indicated concentrations of angiotensin. D, Current-voltage relationships for IBa through 1C,77 channels coexpressed with AT1A receptors, before treatment (), 5 min after treatment with 1 µM angiotensin (), and after a 10-min perfusion with bath medium (), obtained using the same oocyte as in B and C (). Experiments were performed at room temperature (~21°).

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of IBa in X. laevis oocytes by angiotensin depends on the expression of the 1C subunit of Ca2+ channel and the AT1A receptor Oocytes were injected with mRNAs coding for auxiliary 1 and 2 subunits of the Ca2+ channel, mixed with the indicated mRNAs. IBa traces were elicited by 250-msec test pulses to +20 mV from a holding voltage of 90 mV. Average amplitudes of IBa were measured before (control) and 5 min after application of 0.5-10 µM angiotensin. In some experiments, oocytes were injected 30-60 min before measurements with 50 nl of 5 µg/ml PTX, 10 mM GDPS, 10 µM heparin, or 94 mM Cs4BAPTA or were incubated overnight with 10 nM thapsigargin.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Failure of angiotensin to suppress IBa through 1C,86 channels coexpressed with AT1A receptors. A, Representative traces of IBa elicited by stepwise depolarization to +20 mV, from a holding potential of 90 mV, recorded before () and 5 min after () application of 1 µM angiotensin. B, Time dependence of the effect of 1 µM angiotensin added to the external Ba2+ solution (arrow) on IBa through the 1C,86 channels. C, Averaged current-voltage relationships (n = 3) determined before () and 5 min after () application of 1 µM angiotensin. In B and C, the amplitudes of IBa were normalized to maximal IBa in the absence of angiotensin.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   A, Concentration-response relationship for the angiotensin effect. Angiotensin, at the indicated concentrations, was applied to an oocyte coexpressing 1C,77 channels and AT1A receptors. IBa was measured at +20 mV, after 5 min of equilibration. The averaged concentration dependence clearly shows saturation of the effect at 1 µM angiotensin. The curve was normalized to the maximal effect and then fit by the function I = 1/{1 + (IC50/[Ang])n}, where I is the normalized IBa amplitude, IC50 is the concentration of angiotensin producing 50% inhibition of IBa, [Ang] is the concentration of angiotensin in the bath solution, and n is the Hill coefficient. The regression coefficient was 0.992. Values are means ± standard errors of four oocytes. B, Time course of the effect of 1 µM losartan and/or 1 µM angiotensin on peak IBa evoked by stepwise depolarization to +20 mV from a holding potential of 90 mV, in an oocyte expressing 1C,77 channels and AT1A receptors. Horizontal bars, times at which losartan and/or angiotensin was applied to the oocyte. Inset, traces of IBa recorded at 1 (), 4 (), 8 (), and 15 () min in this experiment.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Time course of the development of angiotensin effects on IBa and ICl(Ca) in oocytes coexpressing AT1A receptors and 1C,77 channels. Lower, time dependence of the effect of angiotensin on the holding current, ICl(Ca) (), measured at 90 mV and on IBa () measured at +20 mV. Arrow, time when 1 µM angiotensin was applied to the oocyte. Upper, traces of IBa recorded at the times indicated (numbers in lower).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Dependence of the reversibility of the angiotensin effect on the loading of intracellular Ca2+ stores. Lower, time course of the effect of 1 µM angiotensin applied (arrows 1) to an oocyte bathed in 40 mM Ba2+-containing solution. Test pulses to +20 mV were applied every 30 sec. Arrows 0, washout of angiotensin. The suppressive effect of angiotensin was fully reversed in the oocyte, but washout for 10 min in Ba2+-containing solution did not restore the angiotensin response. Horizontal bar, incubation of the oocyte in normal Barth's solution containing 2 mM Ca2+. Reapplication of angiotensin even after 5 min of incubation in Ca2+-containing Barth's solution restored the angiotensin effect. Upper, traces of IBa recorded at the indicated times.

Angiotensin activates a transient I_Cl. The rapid application of the hormone in Cl-free solutions was often but not always accompanied by activation of a large, transient, inward current lasting ~2 min. The activation of this inward holding current, measured at 90 mV in Clfree extracellular solution (Fig. 4, lower), preceded the decrease in I_Ba. This current had properties similar to those previously identified (Hartzell, 1996; Gomez-Hernandez et al., 1997) for I_Cl(Ca). During the activation of I_Cl, I_Ba often exhibited decreased inactivation kinetics, producing large, slowly deactivating, tail currents (Fig. 4, upper, traces 2 and 3). Interestingly, the angiotensin-induced, transient suppression of I_Ba outlasted the activation of I_Cl(Ca) by 2-3 min (Fig. 4), suggesting either different affinities of Ca²⁺ channels and Ca²⁺-activated Cl channels for Ca²⁺ or differences in the spatial distribution of the two channels with respect to the intracellular Ca²⁺ pools. Lower affinity of I_Cl(Ca) for activation by Ca²⁺, compared with Ca²⁺-induced inactivation of Ca²⁺ channels, and variations in the Ca²⁺ contents of intracellular Ca²⁺ pools of the oocytes might be partly responsible for the variations in the magnitude of I_Cl in different oocytes.

The IP₃/Ca²⁺ signaling pathway is involved in channel regulation by angiotensin. Ca²⁺ stores in X. laevis oocytes are known to be regulated through the activation of IP₃-sensitive Ca²⁺ release channels (Berridge and Irvine, 1989; Putney et al., 1989). These channels are thought to be involved in receptor-mediated Ca²⁺ signaling, and their activation is known to evoke I_Cl(Ca) in oocytes (Yao and Parker, 1993; Hartzell, 1996). Consistent with this idea, in oocytes bathed in Barth's solution and expressing only AT_1A receptors, a transient (2-3-min) I_Cl was activated upon rapid application of angiotensin (data not shown). To further characterize the steps in the regulation of recombinant _1C channels by AT_1A receptors, when coexpressed in oocytes, we probed the various steps of the IP₃-mediated Ca²⁺ signaling cascade by inhibiting the G proteins, blocking the IP₃ receptor, and interfering with the rise in intracellular Ca²⁺ levels.

Release of intracellular Ca²⁺ mediates the angiotensin-induced effects. The depletion of intracellular Ca²⁺ stores by overnight incubation of oocytes with 10 nM thapsigargin (Thastrup et al., 1990) completely abolished the effect of 1 µM angiotensin on I_Ba (Fig. 6, A and B). No significant difference in the amplitude of I_Ba in control and thapsigargin-incubated oocytes was observed (Table 1). Similarly, oocytes injected with Ca²⁺ buffers failed to respond to angiotensin. Fig. 6, C and D, shows data recorded from an oocyte that was injected with 50 nl of 94 mM Cs₄BAPTA solution 30 min before measurements of I_Ba. The data (n = 4) showed that signaling between _1C,77 channels and AT_1A receptors in response to 0.1-1 µM angiotensin was completely suppressed.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Molecular steps in the modulation of IBa by angiotensin. Two sets of pairs of averaged current-voltage relationships (A, C, and E) and time-dependent relationships for the angiotensin effect (B, D, and F), as well as representative current traces, were recorded before (, ) and 5 min after (, ) application of 1 µM angiotensin. Arrows, times at which angiotensin (ATII) was applied. A and B, Examination of the role of intracellular Ca2+ release. Oocytes were incubated overnight with 10 nM thapsigargin (A, , , n = 3; B, ) or injected 30-60 min before measurements with 50 nl of 94 mM Cs4BAPTA (A, , , n = 6; B, ). C and D, Examination of the role of G proteins. Oocytes were injected 30-60 min before measurements with 50 nl of 10 mM GDPS (C, , , n = 4; D, ) or 5 µg/ml PTX (C, , , n = 5; D, ). E and F, Examination of the involvement of IP3 receptors. Oocytes were injected 30-60 min before measurements with 50 nl of 10 µM heparin (molecular weight, 3000) (E, , , n = 4; F, ). We also show the response of a control oocyte, coexpressing 1C,77 channels and AT1A receptors (E, , , n = 6; F, ), to angiotensin before the interventions described in A-F.

PTX-sensitive G proteins and IP₃ receptors mediate the angiotensin-induced effects. AT_1A receptors in mammalian cells are known to be coupled to G proteins (Lu et al., 1996). In X. laevis oocytes coexpressing AT_1A receptors and Ca²⁺ channels, we probed the functional manifestation of G protein coupling. In oocytes that had been preinjected with 50-100 nl of GDPS (10 mM), angiotensin (1 µM) failed to produce significant inhibitory effects on I_Ba (Fig. 6, C and D; Table 1). Because the effect of angiotensin was also blocked in parallel experiments with microinjection of 50-100 nl of PTX (5 µg/ml) (Fig. 6, C and D; Table 1), the coupling between the recombinant AT_1A receptors and _1C,77 Ca²⁺ channels seemed to be mediated through the activation of endogenous G proteins of the G_i type.

A Ca²⁺-insensitive _1C,86 Ca²⁺ channel coexpressed with the AT_1A receptor is not modulated by angiotensin. To examine a molecular motif possibly involved in angiotensin-mediated modulation of Ca²⁺ channels, a recently described Ca²⁺ channel isoform (_1C,86) lacking the Ca²⁺ sensors responsible for Ca²⁺-induced modulation (Soldatov et al., 1997) was coexpressed with AT_1A receptors in X. laevis oocytes. In contrast to the effect of angiotensin on the _1C,77 channel (Figs. 1, 3, and 4), the _1C,86 channel was insensitive to modulation by angiotensin (Table 1). Fig. 2A demonstrates that neither the amplitude nor the kinetics of I_Ba were significantly changed in the presence of angiotensin. There was often a 5-15% increase in the amplitude of I_Ba (Fig. 2B), which resembled the small increase of I_Ba observed in oocytes expressing _1C,77 without the AT_1A receptor (Fig. 1C). Interestingly, the voltage dependence of I_Ba through _1C,86 channels was also reversibly shifted to more positive potentials in the presence of 1 µM angiotensin (Fig. 2C), in a manner similar to that observed for _1C,77 (Fig. 1D). This shift might be the result of additional screening effects of the released Ca²⁺ on the plasma membrane cation-binding sites. The absence of angiotensin effects in oocytes coexpressing _1C,86 with AT_1A receptors suggests that the Ca²⁺ sensors of the Ca²⁺ channel are critical in mediating the suppressive effect of angiotensin on the channel.

	Discussion

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Our results show conclusively that, in the oocyte expression system, human recombinant _1C Ca²⁺ channels can be modulated by angiotensin through AT_1A receptors via the G protein-dependent, IP₃-activated Ca²⁺ release system. Inhibition of any of the key steps in the IP₃-dependent Ca²⁺ signaling pathway, including blockade of AT_1A receptors (by losartan), G proteins (by GDPS or PTX), or IP₃ receptors (by heparin) and depletion of intracellular Ca²⁺ stores (by thapsigargin or BAPTA), eliminated the suppressive effect of angiotensin on the Ca²⁺ channels. The hormone-induced transient increase of the intracellular Ca²⁺ concentration also activated Ca²⁺-dependent Cl channels (Hartzell, 1996; Gomez-Hernandez et al., 1997), which was monitored in our experiments as the transient increase in the holding current at 90 mV (Fig. 4). The Ca²⁺-dependent outward Cl flux (inward I_Cl) seems to produce sufficient increases in membrane conductance to cause slowing of the inactivation kinetics of I_Ba and development of slowly deactivating "tails" (Fig. 4).

It is intriguing to note that, although the hormone suppressed the amplitude of I_Ba by releasing intracellular Ca²⁺, the kinetics of the current was not significantly accelerated (Fig. 1B), as might have been expected from a comparison of Ca²⁺ and Ba²⁺ current traces recorded in the oocyte expression system (e.g., Fig. 2A in the report by Soldatov et al., 1998). One possible explanation for this result is that pore-permeating Ca²⁺ and intracellularly released Ca²⁺ may regulate the _1C channel activity by targeting different molecular sites (Ca²⁺ sensors) associated with the channel. Similar dual modulation of Ca²⁺ channel kinetics by intracellular Ca²⁺ was first observed in dorsal root ganglion neurons (Morad et al., 1988). In that case, photorelease of caged Ca²⁺ (10-50 µM) strongly suppressed the Na⁺ current through the channel, without affecting the kinetics of its inactivation. In support of the idea of dual modulation, we recently reported that a segment (positions 1572-1651) of the cytoplasmic carboxyl-terminal tail of _1C,77 contains two separate Ca²⁺ sensors (molecular determinants for the Ca²⁺-dependent inactivation of the channel) (Soldatov et al., 1998). The identified Ca²⁺ sensors may differentially contribute to the Ca²⁺-induced inactivation of the channel, because they may be selectively targeted by permeating versus cytoplasmic Ca²⁺ because of their specific locations with respect to the pore. Consistent with this idea, the _1C,86 channel, which lacks Ca²⁺ sensors in the carboxyl-terminal tail and does not show Ca²⁺-dependent inactivation, conducts Ca²⁺ and Ba²⁺ with comparable kinetics (Soldatov et al., 1997) and is insensitive to angiotensin-mediated increases in intracellular Ca²⁺ concentrations (Fig. 6; Table 1). Because segment 1572-1651 is the only molecular motif modified in the _1C,86 channel, compared with the _1C,77 channel, we conclude that this locus is largely responsible for the angiotensin-induced modulation of the _1C,77 channel coexpressed with the AT_1A receptor. This modulation takes place when Ba²⁺ is the charge carrier through the channel and is apparently independent of permeation of Ca²⁺ through the pore.

Our data on the differential modulation of Ca²⁺ channels by pore-permeating Ca²⁺ and Ca²⁺ released in the cytosol might indicate critical steps in cross-signaling between the angiotensin receptor and IP₃-gated Ca²⁺ stores. Such dual control adds to the complexity of the mechanisms of cross-talk between Ca²⁺ channels and G protein-coupled receptors and may be of fundamental physiological significance, considering that signaling may take place in confined cellular microdomains.