Functional Expression and Characterization of a Voltage-Gated CaV1.3 ({alpha}1D) Calcium Channel Subunit from an Insulin-Secreting Cell Line

Alexandra Scholze, Tim D. Plant, Annette C. Dolphin and Bernd Nürnberg

Institut für Pharmakologie (A.S., T.D.P., B.N.) Freie Universität Berlin 14195 Berlin, Germany
Department of Pharmacology University College London (A.C.D.) WC1E 6BT, United Kingdom
Abteilung für Pharmakologie und Toxikologie (B.N.) Universität Ulm 89081 Ulm, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type {alpha}1-subunits in pancreatic ß-cells is of the neuroendocrine subtype (CaV1.3 or {alpha}1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or {alpha}1C-a) after expression together with {alpha}2{delta}- and ß3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound Gi/Go-coupled GPCRs whereas a neuronal CaV2.2 (or {alpha}1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by Gi/Go-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Voltage-dependent calcium channels play a central role in the regulation of insulin secretion by pancreatic ß-cells. However, the subtypes of calcium channels involved, their particular contribution to the secretion process, and their regulation by glucose and by transmitter substances are still controversial points. This is due, in part, to the fact that studies have been performed on native ß-cells from different species and also on various insulin-secreting cell lines, e.g. HIT-cells and RINm5F cells.

Using molecular biological approaches, various pore-forming calcium channel {alpha}1-subunits (CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3, and CaV3.1) have been identified in insulin-secreting cells and pancreatic tissue (1, 2, 3, 4, 5). In electrophysiological studies, channel currents with the pharmacological and functional properties known for these subunits have also been reported. Thus, high-voltage-activated (HVA), dihydropyridine (DHP)-sensitive channels (6), corresponding to the CaV1.2 or CaV1.3 subunits, {omega}-conotoxin GVIA-sensitive channels (7, 8), corresponding to the CaV2.2 subunit, and {omega}-agatoxin IVA-sensitive channels (5), corresponding to the CaV2.1 subunit, have been described. Low-voltage-activated currents, which would correspond to the CaV3.1 subunit that has been found in the INS-1 cell line, have also been observed in several insulin-secreting cell types (4).

Taken together, insulin-secreting cells express multiple voltage-gated calcium channel subtypes, making it difficult to clarify the properties of a certain channel type in these cells. A common approach to address this problem is the use of cellular systems overexpressing the proteins of interest. This approach has already been extensively used to study the properties of channels formed by most of the calcium channel {alpha}1-subunits [e.g. CaV1.2 (9, 10), CaV2.1 (11), CaV2.2 (9, 12, 13), CaV2.3 (11, 14); CaV3.1 (15, 16)].

In the case of the CaV1.3 subunit, however, which is thought to play a major role in the regulation of insulin secretion, few data are available (17, 18), although several splice variants were cloned (17, 18, 19, 20). This subunit, in combination with the auxiliary subunits {alpha}2{delta} and ß, forms an HVA-calcium channel of the L-type (for review see Refs. 21, 22, 23). Its occurrence is not restricted to the pancreas. Isoforms of the CaV1.3 subunit have been identified by RT-PCR, antibodies, or Northern blot analysis in brain, heart, kidney, adrenal gland, and osteoblasts (24, 25, 26). CaV1.3 is thought to comprise the major fraction of L-type calcium current in neurosecretory and neuronal cells (Refs. 22, 27 but see also Ref. 25).

In pancreatic ß-cells the majority of L-type calcium channel mRNA codes for CaV1.3 subunits (2, 28). So far, there is only a single brief report on the functional expression of a CaV1.3 channel cloned from an insulin secreting cell line [RINm5F (17)]. Hence, data on the functional properties and regulation of these neuroendocrine CaV1.3-derived pore-forming calcium channel subunits are still lacking. This prompted us to study the CaV1.3 isoform cloned from the insulin-secreting HIT-T15 cell line (19), after coexpression with the {alpha}2{delta} and ß3 subunits in Xenopus oocytes. Here, we provide the first description of the biophysical properties of this isoform and the first description of receptor regulation of a heterologously expressed pancreatic CaV1.3 channel.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calcium Channel Expression
To examine the functional properties of calcium channels, we injected {alpha}1 subunits encoding the neuroendocrine (CaV1.3), cardiac (CaV1.2a), or neuronal (CaV2.2) calcium channel together with the {alpha}2{delta} and ß3 subunits into oocytes. The resulting Ba2+ inward current amplitudes in subsequent voltage-clamp measurements were highly variable (Fig. 1AGo). Differences were considerable between individual donor toads, but much less within a batch of oocytes from the same animal. Uninjected oocytes showed either no detectable native calcium channel current, or inward currents with amplitudes of up to 50 nA. In oocyte batches from most donor animals, endogenous oocyte calcium channel currents were enhanced by exogenous {alpha}2{delta} and ß subunits, as described previously (29). The resulting currents were often of considerable amplitude (see Fig. 1AGo), in agreement with one previous study (29), but in contrast to others (18, 30). Hereafter, the ß/{alpha}2{delta}-stimulated endogenous oocyte calcium channel is referred to as the endogenous calcium channel. To assess the effects on endogenous channels, all types of experiments on inward Ba2+ currents were also performed in cells injected with cRNA for the {alpha}2{delta} and ß subunits. To minimize the contribution of currents through the endogenous channel, the amplitudes of currents through these channels were compared with those from cells injected with cRNA for the respective complete exogenous channel. Only those oocyte batches were used, in which a very large relative proportion (at least 80%) of total current was contributed by the exogenous channel subtype. Up to 20% of endogenous current was present in some cases when examining the CaV1.3 channel, since the amplitude of currents in oocytes expressing this channel was in general smaller than that in those expressing the cardiac CaV1.2a or neuronal CaV2.2 subunits (compare Fig. 1AGo). These latter two {alpha}1 subunits produced large currents, which were usually measurable on the second day after cRNA injection. In contrast, expression of the neuroendocrine CaV1.3 calcium channel exceeded expression of the endogenous channel to a sufficient extent for measurement only after 4–6 days.



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Figure 1. Current Amplitudes, Average I-V Relationship, and Trace Examples of the Channels Examined

A, Maximum current amplitudes for endogenous oocyte (n = 69), the neuroendocrine CaV1.3 (n = 74), and the cardiac CaV1.2a (n = 38) calcium channel current from six donor frogs. B, The average I-V relationship for a) the neuroendocrine CaV1.3 (n = 17), b) the cardiac CaV1.2a (n = 17), and c) the endogenous oocyte (n = 14) calcium channel. Individual I-V curves were normalized to the maximum current, averaged, and then fitted with equation 1Go yielding V1/2 = -9.8 mV for panel Ba, -6.7 mV for panel Bb, and 7.0 mV for panel Bc, respectively. C, In each case representative traces from voltage steps to -30, -20, -10, 0, 10, and 20 mV are shown. The examples in panels Ca–Cc correspond to panel B.

 
When expressed without the auxiliary subunits {alpha}2{delta} and ß (n = 8), the neuroendocrine CaV1.3 subunit did not produce inward currents exceeding those of noninjected oocytes, in contrast to the CaV1.2a subunit (our unpublished data and Ref. 31). Currents of larger amplitude than those in control cells were also not observed when only {alpha}2{delta} was expressed together with CaV1.3. In contrast, when the ß3 subunit was coexpressed with CaV1.3 without {alpha}2{delta}, the resulting current amplitudes were 22–50% of the corresponding CaV1.3/ß3/{alpha}2{delta} currents. In one batch of oocytes that did not display measurable endogenous calcium channel currents after injection of exogenous auxiliary subunits, the following maximum inward current amplitudes were obtained: CaV1.3: 0 nA (n = 4), CaV1.3/ß3: -152 ± 29 nA (n = 6), CaV1.3/ß3/{alpha}2{delta}: -586 ± 141 nA (n = 6).

Activation of Ba2+ Currents
Analysis of the current-voltage (I-V) relationships for the neuroendocrine CaV1.3 and the cardiac CaV1.2a channels (Fig. 1BGo) revealed significant differences with respect to the half-maximal activation parameters (V1/2; P < 0.05), which were -9.6 ± 1.2 mV (n = 17) and -6.1 ± 1.2 mV (n = 17), but not for the slope factors (k), which were 7.5 ± 0.2 mV and 7.3 ± 0.4 mV, respectively. The maximum inward current for the neuroendocrine CaV1.3 channel occurred at more negative potentials (2.7 ± 1.1 mV; n = 14) than for the cardiac CaV1.2a channel (6.4 ± 1.1 mV; P < 0.05). The I-V relation of the endogenous calcium channel showed a V1/2 that was significantly (P < 0.001) shifted to depolarized potentials compared with the CaV1.3 and the CaV1.2a channels. Half-maximal activation occurred at 9.4 ± 1.1 mV, and the slope factor (k) was estimated to be 7.5 ± 0.4 mV. The maximum inward current was at 18.8 ± 1.0 mV, a value comparable to those published previously (10, 18, 31).

It is obvious from the current traces in Fig. 1CGo that the activation of the cardiac CaV1.2a channel is slower than that of the neuroendocrine CaV1.3 or endogenous counterparts. The rising phase of currents elicited by depolarizing voltage steps was quantified by determining the time necessary to reach 90% of peak current [90% time-to-peak (ttp)] in each case (Fig. 2AGo). The cardiac CaV1.2a calcium channel showed a significantly (P < 0.05) slower activation compared with the neuroendocrine CaV1.3 and the endogenous channel over the voltage range between -10 and +20 mV. The activation times for the neuroendocrine CaV1.3 and the endogenous oocyte channels were only significantly different (P < 0.05) at -10 mV. In addition, a voltage dependence of the values for 90% ttp was observed for all three channels with values decreasing with increasing depolarization. This decrease with potential was, however, only significant (P < 0.05) for the neuroendocrine CaV1.3 and the cardiac CaV1.2a channels, when the values of 90% ttp at the different voltages were compared for each individual subtype.



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Figure 2. Time Dependence of Calcium Channel Activation, Current Decay, and Steady-State Inactivation

A, Time-to-90% peak current (90% ttp) during voltage pulses to the indicated potentials for the neuroendocrine CaV1.3 (n = 5), the cardiac CaV1.2a (n = 5), and the endogenous oocyte (n = 5) calcium channel. B, Channel inactivation during 1,500 msec voltage steps to the maximum of the individual I-V curve. Traces were normalized to the maximum current and a representative example is shown for each case. C, Steady-state inactivation data were deduced from peak currents at 0 mV reached after a prepulse to the indicated potentials. Data were pooled from experiments with prepulse lengths between 10 and 90 sec; no significant differences were observed between the differing protocols. The prepulse and the test pulse were separated by a 10- to 20-msec interval at -80 mV. Individual inactivation curves were normalized to the maximum current, averaged, and fitted, yielding a half-maximal inactivation at -37.1 mV for the CaV1.3 ({square}; n = 24), -24.7 mV for the cardiac CaV1.2a ({circ}; n = 20), and -38.5 mV for the endogenous ({blacktriangledown}; n = 7) calcium channel.

 
Inactivation of Ba2+ Currents
From the current traces in Figs. 1CGo and 2BGo, it is clear that the rate of inactivation of calcium channel currents differs for the neuroendocrine CaV1.3, cardiac CaV1.2a, and endogenous channel subtypes. When the current decay during longer depolarizing voltage steps was analyzed in detail, it was found to be best fitted by a monoexponential function (eq 4) when pulses of 1,500 msec were used. Currents during longer pulses (5 sec) were better fitted by a biexponential function (eq 5Go, values in Table 1Go). The neuroendocrine CaV1.3 and the cardiac CaV1.2a channels differed significantly, both in their {tau}1 (P < 0.005) and {tau}2 (P < 0.005) values and in the ratio of the amplitudes of the fast and the slow current components (A1/A2, P < 0.001). The endogenous channel inactivated most rapidly, but differed significantly (P < 0.05) from the neuroendocrine CaV1.3 channel only in the {tau}1 value for the fast current component.


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Table 1. Values from Biexponential Fitting of Current Decay in Calcium Channels

 
Comparing fits of a monoexponential function to currents during 1,500-msec pulses to the maximum of the individual I-V curve (see Fig. 2BGo), {tau} was 546 ± 23 msec (n = 17) for the neuroendocrine CaV1.3 and 972 ± 126 msec (n = 9) for the cardiac CaV1.2a calcium channel, values that were significantly different (P < 0.001; see also Fig. 2BGo). Since endogenous oocyte Ca2+-activated Cl- conductances may obscure the measurement of inactivation of calcium channels, additional experiments were performed on oocytes with the calcium chelator BAPTA [1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium] present inside the cell. The currents during 1,500-msec pulses to the maximum of the individual I-V curve fitted with a monoexponential function, {tau} was 530 ± 15 msec (n = 26) for the neuroendocrine CaV1.3 and 966 ± 88 msec (n = 14) for the cardiac CaV1.2a calcium channel, values that were significantly different (P < 0.001). Hence, the rate of inactivation of CaV1.2a channels was significantly different from inactivation of CaV1.3 channels, both in the presence and absence of the intracellular Ca2+ chelator BAPTA.

Potential Dependence of Ba2+ Current Inactivation
The steady-state inactivation was measured with a two-step protocol. The inactivation curves are shown in Fig. 2CGo. Their analysis indicated that the cardiac CaV1.2a and the neuroendocrine CaV1.3 channels differ significantly (P < 0.001) in their half-maximal inactivation, with respective V1/2 values of -22.7 ± 1.7 (n = 20) and -36.1 ± 0.9 mV (n = 24). Values for the endogenous calcium channel (V1/2 = -35.0 ± 3.5 mV; n = 7) differed significantly from both other channels (P = 0.001). The slope factor (k) was almost identical for all three channels (neuroendocrine CaV1.3: 12.7 ± 0.3 mV; cardiac CaV1.2a: 14.0 ± 0.7 mV; and endogenous: 14.4 ± 1.2 mV).

In addition, we analyzed steady-state inactivation in the presence of intracellular BAPTA. The results also showed a significant difference (P < 0.001) between the cardiac CaV1.2a channel (V1/2 = -20.5 ± 2.3 mV; n = 15) and the neuroendocrine CaV1.3 channel (V1/2 = -30.9 ± 1.2 mV; n = 17).

Current Characterization by Blocking Agents
HVA calcium currents in native ß-cells have been shown to be blocked completely by 200 µM Cd2+ (8). Addition of 200 µM Cd2+ to the bath solution abolished currents through the CaV1.3/ß3/{alpha}2{delta} (n = 6) as well as the CaV1.2a/ß3/{alpha}2{delta} (n = 4) calcium channels.

An important distinguishing feature for L-type calcium channels is their sensitivity to DHPs. We therefore compared the DHP sensitivity of the neuroendocrine CaV1.3, cardiac CaV1.2a, and endogenous channels. Using (+)isradipine (Fig. 3Go), we found a 50% inhibition of the DHP-sensitive fraction of the current at a concentration of 43 nM for the cardiac CaV1.2a channel and a similar value of 57 nM for the neuroendocrine CaV1.3 channel. The endogenous current was not inhibited by (+)isradipine at concentrations up to 3 µM (n = 7), as also shown in other studies (31, 32).



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Figure 3. Comparison of Dihydropyridine Sensitivity of Calcium Channels Composed of CaV1.3 and CaV1.2a Subunits

A, Peak currents within a 50-msec test pulse to 0 mV and an interpulse interval of 30 sec were measured. The (+)isradipine stock was diluted in the bath solution to the indicated concentrations and applied. Data points are quotients of the current amplitude before (+)isradipine application and the point of maximal inhibition (n = 3–5 for each point). Curve fitting (eq 6Go) revealed an IC50 of 57 nM for the CaV1.3 ({blacksquare}) and 43 nM for the cardiac CaV1.2a ({circ}) calcium channel. B, Example of isradipine inhibition for both channels close to the IC50 (neuroendocrine CaV1.3, Ba; cardiac CaV1.2a, Bb). A control trace and one of maximal inhibition during isradipine application are shown for each case.

 
Regulation of the Expressed Calcium Channels
Previously, we and others have found a G protein-dependent inhibition of L-type calcium channels in neuroendocrine cells (7, 33, 34) resembling membrane-delimited inhibition of calcium channel {alpha}1A,B,E-subunits by Gß{gamma}-complexes (12, 35, 36). Furthermore, the neuroendocrine CaV1.3 calcium channel subtype, in particular, has been proposed to be affected by this inhibition (for review see Ref. 22). This prompted us to examine the regulation of the neuroendocrine CaV1.3 channel from HIT-T15 cells in oocytes coinjected with the Gi/Go-coupled µ-opioid receptor. In our hands, this receptor was able to couple to endogenous phospholipase C in oocytes, as indicated by the stimulation of calcium-dependent chloride currents (n = 14) after receptor activation with the selective µ-opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin) in oocytes bathed in modified Ringer. Furthermore, we have ascertained that the µ-opioid receptor used in this study couples only to pertussis toxin-sensitive G proteins of the Gi-subfamily regardless of which agonist was used (37). Moreover, as a control for G protein-mediated inhibition in this system, we tested the neuronal (N-type) CaV2.2 (or {alpha}1B) calcium channel. This channel is the classical example of a voltage-dependent calcium channel inhibited by PT-sensitive G proteins (38, 39). As depicted in Fig. 4Go, application of DAMGO to oocytes coexpressing µ-opioid receptors and CaV2.2 channels resulted in a rapid inhibition of inward currents. The concentration of DAMGO necessary to maximally inhibit this channel was determined from the dose-response relation, which revealed an IC50 of 1.1 nM and maximal inhibition with concentrations above 100 nM (n = 3–5). This maximal inhibition was approximately 60%, in accordance with previous studies of this channel when expressed with calcium channel ß subunits (9, 40). In contrast to the neuronal CaV2.2, the neuroendocrine CaV1.3 channel was stimulated by DAMGO at a maximally effective concentration (1 µM). The effect started within a few seconds of agonist application, reached a maximum increase of 406 ± 49 nA (28 ± 2%, n = 20) within 2 min, and was reversible upon washout of the agonist (see Fig. 4Go). To exclude nonspecific, receptor-independent effects, DAMGO was applied at the same concentration to cells injected with cRNA for the neuroendocrine CaV1.3 channel, but not for the µ-opioid receptor. No stimulation of inward current was observed (n = 5) under these conditions or after the application of bath solution without DAMGO to cells expressing the µ-opioid receptor and neuroendocrine CaV1.3 channel (n = 3). Since it was shown by Zamponi et al. (40) that G protein-mediated inhibition can be impaired as a result of channel phosphorylation by protein kinase C (PKC), oocytes were incubated with H7 at a high concentration (100 µM) to inhibit serine/threonine kinase-dependent phosphorylation as completely as possible (Fig. 4BGo). After 5 min (n = 3) or 30 min (n = 2) of preincubation with H7, the increase in current through the neuroendocrine CaV1.3 channel after opioid receptor activation was prevented. However, under these conditions DAMGO still failed to induce calcium channel inhibition (see Fig. 4BGo). We therefore concluded that DAMGO-induced neuroendocrine CaV1.3 calcium channel stimulation involved protein phosphorylation. Stimulation of calcium channel currents in neuroendocrine cells resulting from phosphorylation by protein kinase A (PKA) and PKC has been reported in several studies (41, 42). Since PKA can also be inhibited by H7 (43), we tested the effect of PKA inhibition on receptor-stimulated channel activity. The response of the neuroendocrine CaV1.3 calcium channel to receptor activation in oocytes injected with the PKA-inhibitor Rp-cAMPS (adenosine 3',5'-cyclic phosphorothiolate-Rp) [to calculated final concentrations of 50 µM (n = 4) and 400 µM (n = 5), 15–20 min before experiments], did not significantly differ from that in control cells without Rp-cAMPS (n = 9). Since L-type calcium channels are known to be stimulated by G protein-coupled receptors (GPCRs) in a phosphoinositol-3-kinase (PI3K)- and/or PKC-dependent manner, we tested the effects of the PI3K-inhibitor wortmannin and the PKC-inhibitor bisindolylmaleimide I on calcium current stimulation. Incubation of oocytes with 150 nM and 500 nM wortmannin in the extracellular solution for 20 min did not result in a significant reduction of the DAMGO-induced effect (n = 5). In contrast, preincubation of oocytes with bisindolylmaleimide I (100 nM, 1 h) resulted in a significant reduction of DAMGO-induced calcium current stimulation (P < 0.005; Fig. 4BGo). Furthermore, we used the phorbol ester phorbol myristate acetate (PMA) to directly activate PKC and obviate potential complications due to overlapping inhibitory effects via Gi/o proteins and stimulatory effects via PKC on calcium channel activity. In line with the results obtained with DAMGO, acute application of PMA (100 nM; n = 12) increased the Ba2+ inward current (107.5 ± 13.4 nA, n = 12) significantly within 2.5 min when compared with results from experiments with vehicle only (46.7 ± 11.1 nA, n = 9) in cells overexpressing the neuroendocrine CaV1.3 channel (P < 0.005).



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Figure 4. Channel Regulation via the µ-Opioid Receptor Agonist DAMGO (1 µM)

A, Typical current amplitude measurements representing peak currents at voltage steps to 0 mV are shown for the CaV1.3 ({square}), the endogenous oocyte ({blacktriangledown}), and the neuronal ({blacktriangleup}) calcium channel. The pulse frequency was 0.2 Hz. B, Maximum current changes under DAMGO application (2.5 min) for the neuronal (n = 7), the endogenous (n = 23), and the neuroendocrine CaV1.3 (n = 26) calcium channel. The fourth column shows the DAMGO effect on the neuroendocrine CaV1.3 channel after preincubation with H7 (100 µM; n = 5); the fifth column shows the DAMGO effect on this channel after preincubation with bisindolylmaleimide I (Bis I; 100 nM; n = 5). Both inhibitors were also present during the DAMGO application in the respective experiments.

 
It should be mentioned that the endogenous oocyte calcium channel was also stimulated by DAMGO. Even though this stimulation was pronounced (200 ± 21%), the absolute amplitude of the effect and the kinetics of this stimulation allowed a clear distinction between the responses of the neuroendocrine CaV1.3 and the endogenous channel (see Fig. 4Go). After agonist application, the increase in currents through the endogenous channel started with a delay, reaching a maximum usually during washout of the agonist (see Fig. 4Go). Since no current stimulation occurred in the presence of H7, stimulation of the endogenous channel probably results from protein phosphorylation. This observation is also in agreement with those that showed that the amplitude of currents through the endogenous oocyte calcium channel was increased by PMA (Ref. 32 and our unpublished data).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Here we describe the successful expression of a previously cloned neuroendocrine L-type calcium channel consisting of the CaV1.3 subunit from insulin-secreting cells together with the ß3 and {alpha}2{delta} subunits (19). Channels of this subunit combination are likely to occur in native endocrine cells since CaV1.3, ß2, and ß3 have been found to coincide in RINm5F cells (17). Furthermore, studies using in situ hybridization and immunostaining found a colocalization of CaV1.3 and ß3 in several brain areas (44).

Notably, currents through this neuroendocrine channel were not observed upon expression of the CaV1.3 subunit alone or in combination with {alpha}2{delta}. Hence, its association with at least ß subunits seem to be essential for functional expression, as also reported for two other CaV1.3 isoforms (17, 18). Nevertheless, upon coexpression of {alpha}2{delta} with the CaV1.3/ß3 subunits, the amplitude of neuroendocrine CaV1.3 channel currents was increased by 2- to 5-fold in the present study. Similar effects of the {alpha}2{delta} subunit on the CaV2.1/ß combination were reported by de Waard and Campbell (30), who found a 2.5-fold current increase. An even more pronounced effect (~15-fold stimulation) was also reported (10), when CaV1.2a/ß/{alpha}2{delta} channels were compared with CaV1.2a/ß channels.

The neuroendocrine CaV1.3 channel examined here yielded, on average, relatively low current amplitudes as compared with channels formed by the CaV2.2 and CaV1.2a subunits in the same expression system. Furthermore, the time interval after cRNA injection that was necessary to obtain currents of sufficient amplitude for kinetic analysis was longer than that required for the other {alpha}1 subunits. Reasons for this observation may be inherent to the CaV1.3 subunit itself. As suggested by Hofmann et al. (23), one reason may be the need for additional, possibly still unidentified, auxiliary subunits. Another possible cause could be the necessity for specific interactions with additional proteins, as described for syntaxin and the CaV1.3a subunit in pancreatic ß-cells (27).

When the I-V curves for the neuroendocrine CaV1.3 channel used in the present study were analyzed, the maximum inward currents were found to occur at potentials between 0 and +10 mV. A similar value of 0 mV was reported by Williams et al. (18) for a neuronal CaV1.3/ß2/{alpha}2{delta} subunit combination expressed in Xenopus oocytes. A particularly interesting native cell for comparison in this respect is the rat pinealocyte, which has been shown to contain CaV1.3 as the sole calcium channel {alpha}1 subunit, together with the ß2 and ß4 subunits. The minimum of the I-V curve in these cells is also between 0 and 10 mV at similar Ba2+ concentrations (26). When comparing the potential dependence of the currents, it should be kept in mind that the ß subunit type also influences this property of calcium channels and, thus, the position of the I-V curve (23). The comparison of the I-V minima, which we performed here, seems to be applicable, since at least in the case of the CaV2.1 subunit, ß2 or ß3, the isoform used in the present study, had a similar influence on the potential dependence of the currents (30).

During prolonged depolarizations, the neuroendocrine CaV1.3 channel showed slow inactivation with Ba2+ as the charge carrier. Inactivation was, however, on average twice as fast for the neuroendocrine CaV1.3 than for the cardiac CaV1.2a channel. The voltage dependence of inactivation also differed significantly between these two L-type channels, with the neuroendocrine CaV1.3 subtype inactivating at more negative membrane potentials. In mouse pancreatic ß-cells, where the majority of the calcium current has been reported to be DHP-sensitive, inactivation during short voltage pulses was shown to be almost purely Ca2+-dependent (3). Other authors also reported a strong calcium-dependence in mouse pancreatic ß-cells (45), but detected an additional slower voltage-dependent component of HVA current inactivation when using longer depolarizing pulses and Ba2+ as the charge carrier. The inactivation of the neuroendocrine CaV1.3 channel in our study, also using Ba2+ as the charge carrier, could partially reflect this component. Although the Ca2+-dependent component dominates inactivation in ß-cells, slower voltage-dependent inactivation could play a role in the regulation of calcium entry during slow wave depolarization.

Sensitivity to DHPs is a property that has frequently been used to distinguish the role of different calcium channel subtypes in the regulation of insulin secretion and the signal transduction processes in insulin-secreting cells (6, 46, 47). Comparison of the DHP sensitivity of the neuroendocrine CaV1.3 and the cardiac CaV1.2a channel revealed similar IC50 values for (+)isradipine. Therefore, this DHP does not discriminate between these two channel isoforms.

Regulation of L-type calcium channels in insulin-secreting cells, either directly by heterotrimeric G proteins or through second messengers, is a matter of intense research (6, 42). For instance, GPCRs have been reported to inhibit L-type calcium currents in insulin-secreting RINm5F cells via PT-sensitive G proteins (6, 7, 47). Since heterologously expressed CaV1.2 ({alpha}1C) calcium channels are not a target for G protein-dependent inhibition (9, 35), CaV1.3 ({alpha}1D) isoforms are L-type channel candidates for inhibition by G proteins. However, in the present study, activation of PT-sensitive G proteins by µ-opioid receptors did not result in inhibition of the neuroendocrine calcium channel (CaV1.3 or {alpha}1D) despite clear inhibition of the neuronal calcium channel (CaV2.2 or {alpha}1B) under identical experimental conditions. In this context it should be noted that the CaV2.2 contains the known Gß{gamma}-binding motif QXXER present in the intracellular loop between repeats I and II. Resequencing of the CaV1.3 by us revealed the presence of a QXXEE-amino acid motif in the loop I-II sequence, which has been found in various L-type calcium channels including a neuronal isoform known not to be directly inhibited by Gß{gamma} (23, 39, 48).

In contrast, we found that currents through this CaV1.3 channel were increased after receptor activation. The stimulation of the CaV1.3 channel is clearly distinguishable from that of the endogenous channel both in time course and amplitude. The finding that the specific kinase inhibitors H7 and bisindolylmaleimide I abolished this receptor-induced current stimulation is mechanistically relevant and points to the involvement of a kinase. Stimulation of voltage-dependent calcium channels by PKC or PKA is well established (23). H7 inhibits both kinases with IC50 values in a comparable range whereas bisindolylmaleimide I is a potent inhibitor of PKC (43, 49). Furthermore, we recently showed that smooth muscle L-type calcium channels are activated by Gß{gamma}-subunits via PKC- and PI3K-dependent pathways (50, 51). However, for GPCR-controlled regulation of the CaV1.3 channel examined in this study, the involvement of PI3K is unlikely, since the specific PI3K inhibitor wortmannin did not affect receptor-induced stimulation of the CaV1.3 channel at a concentration known to block class I PI3Ks (data not shown). Also, the involvement of PKA in transmitting the stimulatory activity from the receptor to the CaV1.3 channel is unlikely based on independent and complementary experimental approaches. First, we ascertained that the human µ-opioid receptor used in the present study couples exclusively to members of the Gi/Go family but not to Gs proteins, which are upstream activators of adenylyl cyclases and PKA (37). This selective coupling between the receptor and G proteins was independent of the ligand used. Second, a stimulation by the downstream effector PKA was ruled out by using the PKA inhibitor Rp-cAMPS. In contrast, several considerations argue for an involvement of PKC in stimulation of the CaV1.3 channel by the Gi/Go-coupled µ-opioid receptor. It is well documented that PKC is activated by PT-sensitive GPCRs via Gi/Go proteins and phospholipase C (for review see Ref. 52). Accordingly, we confirmed that the µ-opioid receptor expressed in Xenopus oocytes was able to activate a calcium-dependent chloride current under appropriate experimental conditions. This stimulatory effect in Xenopus oocytes is an established read-out system to detect coupling between receptors and endogenous phospholipase C, the key regulator of PKC (53). We also confirmed in a more direct fashion the ability of PKC to stimulate the CaV1.3 channel using the PKC activator PMA. Accordingly, recent studies described a stimulation of L-type calcium channel currents due to phosphorylation by PKC in the insulin-secreting cell lines HIT-T15 and RINm5F (42, 46).

In conclusion, the present study provides the first detailed description of a CaV1.3 (L-type {alpha}1D) calcium channel subunit cloned from an insulin-producing cell line. The importance of L-type channels for the exocytotic process has been stressed by several authors (5, 54). It is therefore interesting that the neuroendocrine CaV1.3 and the cardiac CaV1.2a L-type channel subtypes, which are often coexpressed, as in insulin-secreting cells (19, 20), display significant differences with respect to specific biophysical properties. Differences in the kinetics of channel activation and inactivation suggest that the two channels may have different roles during depolarization-induced calcium influx in ß-cells and consequently in insulin secretion. Most interestingly, this CaV1.3 isoform is stimulated by GPCRs in a protein kinase-dependent manner.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
DAMGO was purchased from RBI (Natick, MA); H7 [(±)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine, 2 HCl] was from Tocris Cookson (Bristol, U.K.); and Rp-cAMPS was obtained from Calbiochem (Bad Soden, Germany). Stocks of those substances were prepared using double distilled water and stored at -20 C. The dihydropyridine (+)isradipine (R(+)-enantiomer of PN200–110) was a kind gift from Sandoz Pharmaceuticals Corp. (Basle, Switzerland). The stock solution of this substance was prepared with dimethylsulfoxide (DMSO). Collagenase type 1A and wortmannin were from Sigma-Aldrich Corp. (Deisenhofen, Germany). Wortmannin was stored in DMSO in the dark at -20 C. The calcium chelators BAPTA and the membrane permeable derivative BAPTA-AM were dissolved in a Tris-containing buffer (pH 7.2) or in DMSO and stored under N2, respectively. Phorbol-12-myristate-13-acetate (PMA) and bisindolylmalimide I [3-(N-[dimethylamino] propyl-3-indolyl)-4-(3-indolyl) maleimide] were from Sigma (St. Louis, MO), and the stock solutions were prepared with DMSO. All other chemicals were of highest purity available.

Expression Plasmids and Oocyte Preparation
The identity of the CaV1.3 clone was verified by sequencing.1 After the addition of an artificial nucleotide sequence that was constructed to optimize translation, the coding sequence started at position 9 of the original amino acid sequence (19). No other differences were found except amino acid positions 427 and 428 of the original amino acid sequence where we found triplets coding for lysine and glutamine instead of asparagine and glutamate, respectively. Capped cRNA transcripts encoding CaV1.3 [XhoI-linearized/T7 RNA polymerase (19)], CaV1.2a [KpnI/SP6 (10)], CaV2.2 [SalI/SP6 (13)], and {alpha}2{delta}-1, termed {alpha}2{delta} [SalI/SP6 (10)], ß3 [NotI/T7 (55)] calcium channel subunits as well as the µ-opioid receptor [Kas I/T7 (56)] were synthesized from the respective cDNAs using the mMessage mMachine in vitro transcription kit (Ambion, Inc. Austin, TX). Stage V–VI oocytes were surgically removed from adult female Xenopus laevis toads (African Xenopus Facility, Knysa, Republic of South Africa) anesthetized with 3-aminobenzoic acid ethyl ester (0.1% wt/vol) according to protocols approved by state and institutional regulations. The incision was sutured immediately after removal of the oocytes and the animals returned to the tank after recovery from surgery. They were then allowed to recover for at least 6 months before being reused as oocyte donors. Follicular cell-free oocytes were obtained by enzymatic isolation using collagenase type 1A (2 mg/ml) in Ca2+-free Barth’s solution (in mM: NaCl, 88; KCl, 1; NaHCO3, 2.4, MgSO4 , 0.82; HEPES, 10; pH 7.4 with NaOH) for 2 h and then stored in Ca2+-containing Barth’s solution (in mM: NaCl, 88; KCl, 1; NaHCO3, 2.4; MgSO4, 0.82; HEPES, 10; CaCl2, 0.41; Ca(NO3)2, 0.33; pH 7.4 with NaOH) supplemented with 2.5 mM sodium pyruvate, 0.1% wt/vol BSA, and 100 µg/ml gentamycin at 18 C. The oocytes were allowed to recover for 24 h and then injected with the same relative proportions of the appropriate cRNAs at a concentration of 0.1 µg/µl. Using a Nanoject Oocyte Injector (Drummond Scientific Co., Broomall, PA), 50 nl of cRNA mixture were injected per oocyte. Barth’s solution was renewed daily and cells were used on days 2–7 after injection, depending on the measured current amplitudes and the quality of the oocytes.

For clarity, the following synonyms were used for the respective injected subunit combinations: neuroendocrine calcium channel for CaV1.3/ß3/{alpha}2{delta}, cardiac CaV1.2a calcium channel for CaV1.2a/ß3/{alpha}2{delta}, endogenous oocyte calcium channel for ß3/{alpha}2{delta}, and neuronal calcium channel for CaV2.2/ß3/{alpha}2{delta}.

Electrophysiological Recording
Two-electrode voltage-clamp currents were recorded using an Oocyte clamp OC-725A amplifier (Warner Instrument Corp., Hamden, CT). Voltage commands were generated and currents recorded using a personal computer interfaced with an TL-1 DMA Interface (Axon Instruments, Foster City, CA) to the amplifier and using the pClamp version 6 software (Axon Instruments). Microelectrodes were filled with 3 M KCl and had typical resistances of 0.5–2.5 M{Omega}. The bath was connected to the clamp circuit via a 3 M KCl-agar bridge. The bath solution contained in mM: Ba(OH)2, 35, NaOH, 50; HEPES, 10; pH 7.4 with methanesulfonic acid. Ba2+ was used as the charge carrier in all calcium channel experiments. For experiments on calcium-dependent chloride currents, modified frog Ringer (in mM: NaCl, 90; KCl, 2.5; CaCl2, 1.8; HEPES, 10; pH 7.4 with NaOH) was used. Throughout all experiments, including drug applications, the bath was continuously perfused with solution at a rate of 2 ml/min. Oocytes were clamped at a holding potential of -60 mV, in the case of the steady-state inactivation experiments it was -80 mV. Only those recordings were used where currents could be adequately voltage clamped. For all experiments conducted with CaV1.3, CaV1.2a, or CaV2.2-calcium channels, at least five control oocytes injected with a corresponding amount of {alpha}2{delta} and ß3 cRNA were measured on the same day. Only those oocyte batches were used for analysis for which the resulting endogenous calcium current in control oocytes comprised less than 20% of the total inward barium current in oocytes expressing the exogenous channels. In the case of experiments where current kinetics were determined, the maximum endogenous currents were <20% of the total current. Only those experiments were considered for final analysis that showed inward holding currents of less than 10% of the maximum inward current and only minimal fluctuations during the course of the experiment.

Data Analysis and Presentation
Where appropriate, values are given as means ± SE. Statistical significance was evaluated by one-way ANOVA, and individual P values are given. The term n represents the number of oocytes tested. Curve fitting was done by a least-squares minimization method using the following equations. For I-V curves:

(1)
where g is the maximum conductance, Vt the test potential, E the apparent reversal potential, V1/2 the potential of half-activation, and k a slope factor. For steady-state inactivation curves,

(2)
where Vp is the prepulse potential, C a baseline, and I has inactivated by half at the prepulse potential indicated by V1/2 with an e-fold change over k mV. For determination of channel activation from tail currents,

(3)
as in the preceding equation, with Vt the test potential and V1/2 the potential of half-maximal activation. For the analysis of tail currents only well clamped oocytes were selected, and to avoid contamination by the capacity transient, values were taken 4 msec after the voltage step. For monoexponential or biexponential fitting of inactivation during a voltage step,

(5)
respectively, where {tau} is the time (t) at which the current had decreased to 1/e of its initial amplitude, A is the maximal amplitude of the component, and C is the steady-state current levels. Dose-response curves for (+)isradipine were fitted applying

(6)
where f represents the maximal fraction of current responsive to the substance, which was calculated by subtracting the relative contribution of endogenous inward Ba2+ current from total inward current. The IC50 value is the substance concentration resulting in half-maximal current inhibition, and c is the substance concentration tested. The Hill coefficient, n, was set to 1. When normalized data were fitted, the same equations apply for I/Imax.

DAMGO was used to activate the µ-opioid receptor expressed after coinjection of its cRNA. The resulting effect on each current was calculated between the last data point before agonist application and the point of maximal effect. Current run-down was not taken into account.

Note Added in Proof
While this paper was in press, Striessnig and co-workers (57) published biophysical and pharmacological properties of an {alpha}1D subunit from cochlea inner hair cells.


    ACKNOWLEDGMENTS
 
We thank the following for generous gifts of cDNAs: Dr. L. Birnbaumer, (University of California, Los Angeles, CA), CaV1.3; Dr. F. Hofmann (Technische Universität München, Munich, Germany), CaV1.2a, ß3, {alpha}2{delta}; Dr. Y. Fujita (Matsushita Electric Industrial Co., Seika, Japan), CaV2.2; and Dr. L. Emorine (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France), µ-opioid receptor. We thank Dr. Günther Schultz for continuous support. We also thank Drs. A. Zobel, C. Canti, and A. G. Obukhov for introduction into the Xenopus laevis oocyte model, as well as A. Tomschegg for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Bernd Nürnberg, Abteilung für Pharmakologie und Toxikologie, Universität Ulm, Albert-Einstein Allee 11, D-89081 Ulm, Germany. E-mail: bernd.nuernberg{at}medizin.uni-ulm.de

This work was supported by the Deutsche Forschungsgemeinschaft, Royal Society, and Fonds der Chemischen Industrie.

1 The nucleotide sequence data have been submitted to the EMBL Nucleotide Sequence Database under accession number AJ311617. Back

Received for publication June 29, 2000. Revision received March 12, 2001. Accepted for publication April 2, 2001.


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