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J. Biol. Chem., Vol. 276, Issue 42, 38727-38737, October 19, 2001

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Functional Properties of Ca_V1.3 (_1D) L-type Ca²⁺ Channel Splice Variants Expressed by Rat Brain and Neuroendocrine GH₃ Cells^*

Parsa Safa^§, Jim Boulter^¶, and Tim G. Hales

From the  Department of Pharmacology, The George Washington University, Washington, DC 20037, the ^§ Neuroscience Interdepartmental Program, University of California, Los Angeles, California 90095, and the ^¶ Department of Psychiatry and Biobehavioral Sciences, Neuropsychiatric Institute, University of California, Los Angeles, California 90095

Received for publication, April 25, 2001, and in revised form, July 24, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca²⁺ entry through activation of pertussis toxin-sensitive G proteins leading to activation of K⁺ channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca²⁺ channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three ₁ (_1A, _1C, and _1D), four , and one ₂- subunit in rat pituitary GH₃ cells; _1B and _1S transcripts were absent. GH₃ cells express multiple alternatively spliced _1D mRNAs. Many of the _1D transcript variants encode "short" _1D (_1D-S) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other ₁ subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (_1D-L). We cloned and expressed the predominant _1D-S transcript variants in rat brain and GH₃ cells and their _lD-L counterpart in GH₃ cells. Unlike _1A channels, _1D channels exhibited current-voltage relationships similar to those of native GH₃ cell Ca²⁺ channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced _1D transcripts form functional Ca²⁺ channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of _1D-S subunit, is not sufficient to confer sensitivity to inhibitory G proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

All excitable cells express voltage-activated Ca²⁺ channels fulfilling diverse cellular functions. Among other things, Ca²⁺ influx can regulate gene expression and propagate action potentials, and it is a prerequisite for sustained neurotransmitter and hormone release (1, 2). Specialized Ca²⁺ channels have evolved to serve these specific purposes. Ca²⁺ channels native to excitable cells can be classified into five categories (T, L, N, P/Q, and R) on the basis of their unique biophysical and pharmacological properties. The genetic basis for the heterogeneity of Ca²⁺ channels is apparent now that genes have been identified that encode ten ₁ (_1A through _1I, and _1S) subunits, four  subunits and three ₂- dimers (3). Ca²⁺ channel ₁ subunits have been grouped into three families (Ca_V1, 2, and 3) on the basis of their levels of amino acid sequence identity (4). Additional structural heterogeneity occurs through alternative splicing (5-9). Expression of recombinant homomeric ₁ subunits in Xenopus oocytes or cell lines produces functional Ca²⁺ channels with properties reminiscent of those of their naturally expressed counterparts. Although the ₁ subunit is the principle component of the Ca²⁺ channel, containing 24 membrane-spanning regions that form both the channel and the voltage sensor, coexpression of ₁ subunits with  and ₂- subunits produces a greater current density and alters voltage dependence, current amplitude, activation, and inactivation kinetics (10-12).

High voltage-activated P/Q- and N-type Ca²⁺ channels have been extensively studied because of their central role in controlling Ca²⁺ entry into, and thus neurotransmitter release from, neuronal synaptic terminals (13). The activity of these channels is inhibited by the activation of metabotropic receptors that couple to pertussis toxin-sensitive G proteins. This pathway is thought to be an important means of presynaptic inhibition, allowing the feedback control of neurotransmitter release. Following metabotropic receptor activation, subunits liberated from pertussis toxin-sensitive G_i/o protein complexes bind directly to P/Q- and N-type Ca²⁺ channels at their QXXER amino acid motifs making them less "willing" to open when stimulated by an action potential invading the synaptic terminal (14-18). The inhibition is voltage-dependent; prolonged (19) or repetitive (20) depolarization can overcome blockade.

Inhibitory metabotropic receptors can also attenuate the activity of L-type Ca²⁺ channels in several neuroendocrine cells and some neurons (21, 22). In the growth hormone- and prolactin-secreting anterior pituitary GH₃ cell line, activation of either native somatostatin and muscarinic receptors or recombinant opioid receptors leads to inhibition of L-type Ca²⁺ channel activity (23-25). This effect can be prevented by pertussis toxin pretreatment or reversed by strong depolarization, suggesting the involvement of subunits. Voltage-dependent coupling between metabotropic receptors and cloned P/Q- and N-type channels can be reconstituted in recombinant expression systems by expressing _1A or _1B subunits, respectively, with the inclusion of auxiliary  and ₂- subunits. For example, cloned µ opioid receptors couple to Ca²⁺ channels containing _1A or _1B Ca²⁺ channel subunits expressed in Xenopus oocytes (26). By contrast, expression of the _1C subunit forms L-type Ca²⁺ channels that are insensitive to opioid receptor activation.

The prevalence of _1D subunit expression in neuroendocrine cells led to the suggestion that this may be the molecular identity of the G protein-coupled L-type channel (21, 22). Furthermore, the existence of a QXXER amino acid motif in an _1D subunit splice variant led us to further hypothesize that this may be necessary for direct G protein-mediated L-type channel inhibition (27). In this study we used RT-PCR¹ to identify Ca²⁺ channel transcripts in GH₃ cells. In addition to _1A and _1C transcripts, we found numerous alternatively spliced _1D transcripts. The predicted amino acid sequences of the _1D-splice variants terminated with either a long (_1D-L) or a short (_1D-S) C terminus, the latter of which contained a QXXER amino acid motif. We used long range RT-PCR to compare the relative prevalence of alternatively spliced _1D transcripts in GH₃ cells and rat brain and created cDNAs encoding _1D-S and corresponding _1D-L splice variants for electrophysiological analyses. Using these clones we examined the functional properties of several alternatively spliced _1D isoforms and determined whether the QXXER motif is sufficient to confer sensitivity of the _1D subunit to G proteins either activated directly by GTPS or through opioid receptor activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cultures and Transfections-- Control GH₃ cells (ATCC, Manassas, VA) and GH₃ cells were stably transfected with µ- and -opioid receptors (GH₃MORDOR cells) as described previously (25). Both cell lines were grown in Dulbecco's modified Eagle's medium containing 10% v/v calf serum, penicillin (100 units/ml) and streptomycin (100 µg/ml). GH₃MORDOR cells were grown under positive selection conditions with hygromycin (200 µg/ml) and Geneticin (400 µg/ml). Cells were subcultured once each week and seeded into 75-cm² flasks, for maintenance, and into 35-mm diameter dishes for use in electrophysiological experiments. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells stably transfected with either - (HEKDOR and CHODOR cells) or µ- (HEKMOR and CHOMOR cells) opioid receptors, a gift from Dr. C. Evans (Department of Psychiatry, UCLA, Los Angeles, CA), were grown in the medium described above for GH₃ cells under positive selection with Geneticin (400 µg/ml). Cells were subcultured 24 h prior to transfection. cDNAs encoding _1D, _1A provided by Dr. Y. Mori (National Institute for Physiological Sciences, Okazaki, Japan), _2a provided by Dr. E. Perez-Reyes (University of Virginia, Charlottesville, VA), and ₂- provided by Dr. L. Birnbaumer (UCLA) were transiently transfected in combination with green fluorescence protein cDNA into HEK or CHO cells by either the CaPO₄ precipitation or electroporation technique. In some experiments, cells were also transfected with cDNAs encoding G_oA or G_oB provided by Dr. M. Simon (California Institute of Technology, Pasadena, CA), -ARK minigene provided by Dr. R. Lefkowitz (Duke University, Durham, NC), or syntaxin-1A provided by Dr. G. Zamponi (University of Calgary, Calgary, Canada). Cells were incubated in a humid atmosphere of 5% CO₂ and 95% air at 37 °C.

Patch-clamp Recordings-- Green fluorescence protein-expressing transiently transfected cells were located using fluorescence microscopy (Nikon Diaphot, Image Systems Inc., Columbia, MD). The whole-cell patch-clamp (Axopatch 200A amplifier, Axon Instruments Inc., Foster City, CA) technique was used to record Ca²⁺ channel activity with Ba²⁺ as the charge carrier. Cells were initially bathed in a solution containing (in mM) NaCl 140, KCl 4.7, MgCl₂ 1.2, CaCl₂ 2.5, and HEPES 10 (pH 7.2). After establishing the whole-cell configuration, the bath solution was replaced by one comprised of (in mM): NaCl 125, CsCl 5.4, BaCl₂ 10.8, MgCl₂ 1, and HEPES 10. In all cases electrodes contained (in mM): CsCl 120, EGTA 10, MgCl₂ 1, Mg-ATP 3, and HEPES 10 (pH 7.2). The potential difference between the open electrode and the bath ground was zeroed prior to establishing a 1 G resistance seal. The liquid junction potential was negligible, and no compensation was made for its cancellation. Unless otherwise stated voltage-activated Ba²⁺ currents were recorded from cells depolarized from the holding potential of 80 to 0 mV for 80 ms at 10-s intervals. The voltage dependence of current inhibitions induced by opioids or GTPS (300 µM) was investigated using a double-pulse protocol: cells were depolarized from a holding potential of 80 mV to between 50 and 60 mV (10-mV increments, 95-ms duration). The pre-pulse was followed (after 10 ms at 80 mV) by a 10-ms pulse to 0 mV. The effect of a depolarizing prepulse on the current-voltage relationship was investigated by applying a 60-mV pulse for 26 ms between two depolarizing test pulses (from 80 mV to between 50 and 60 mV in 10-mV increments). Time required for recovery from voltage-dependent facilitation was examined by depolarizing cells from 80 to 60 mV followed by 10-140 ms (in 10-ms increments) by a test pulse to 0 mV. Currents were low-pass-filtered at 2 kHz and digitized (Digidata, Axon Instruments Inc., CA) at 10 kHz for storage on the hard drive of a Pentium PC. Tail currents were recorded using Sylgard (Dow Corning Corp., Midland, MI)-coated borosilicate glass (Garner Glass Co., Claremont, CA). Tail currents generated by repolarization to 80 mV following 5.8-ms depolarizations to voltages between 60 and 60 mV were filtered at 10 kHz and digitized at 200 kHz for storage on a PC hard disc. Experiments were performed at room temperature (22-24 °C).

Curve Fitting and Statistics-- Tail currents were fitted by single exponentials using pCLAMP8 software (Axon Instruments Inc, CA). Tail current amplitudes were derived by extrapolating the fit to the point of hyperpolarization. There was no relationship between test pulse potential and the time constant () of the tail currents. The relationship between tail current amplitude and the voltage of depolarization was fitted with a Boltzmann equation of the form,

(Eq. 1)

where I_tail is the tail current amplitude as a percentage of maximum, V is the command voltage, V_1/2 is the voltage for 50% maximal activation, and k is the slope factor.

Concentration-response curves were fitted using the logistic equation,

(Eq. 2)

where I is the Ba²⁺ current amplitude in the presence of a specific concentration of nimodipine expressed as percent control, E_max is the maximal percent inhibition of the current, IC₅₀ is the concentration of nimodipine that had a half-maximal effect, and n is the slope.

Data obtained from the voltage-dependent reversal of Ba²⁺ current inhibitions were fitted with the Boltzmann equation,

(Eq. 3)

where I is the Ba²⁺ current amplitude as a percentage of control current amplitude, F_max is the maximum percent facilitation of the current amplitude, x is the prepulse potential, F₅₀ is the prepulse potential required for half-maximal facilitation, and S is the slope factor. In these experiments control amplitude was the amplitude of the current activated by stepping from 80 to 0 mV after a prepulse to 50 mV.

Data are expressed as means ± S.E., and statistical significance was established using the Student's t test.

Drugs Used-- Pertussis toxin (from Sigma Chemical Co., St. Louis, MO) was applied to the culture medium at a final concentration of 100 ng/ml for at least 24 h before experiments. All other drugs were diluted into the extracellular solution that constantly perfused the recording chamber through gravity feed at a rate of ~5 ml/min. [D-Pen²,D-Pen⁵]enkephalin (DPDPE) was obtained from Peninsula Laboratories (Belmont, CA). GTPS (Life Technologies, Inc., Gaithersburg, MD) was loaded into the recording electrode at a final concentration of 300 µM. Recordings were made ~3 min after achieving the whole-cell configuration. All tissue culture reagents, including antibiotics were obtained from Life Technologies, Inc. (Gaithersburg, MD).

RT-PCR-- RNA was prepared from whole rat brain and GH₃ cells (passage 12) and reverse-transcribed using random hexamers or poly-dT. Specific oligonucleotides were designed for ₁ subunits (_1B-1E, _1S),  subunits (_1-4), and the ₂- subunit. GenBank^TM accession numbers, forward and reverse primers, respectively, are as follows: _1B M92905: 5854-5877, 6427-6450; _1C M67516: 5957-5980, 6491-6514; _1D D38101: 5001-5024, 5475-5500; _1E L15453: 5892-5915, 6436-6459; _1S U31816: 5-28, 821-844; ₁ X61394: 1588-1611, 2053-2076; ₂ M80545: 1642-1665, 2054-2077; ₃ M88751: 1213-1236, 1498-1522; ₄ L02315: 1448-1471, 1736-1759; ₂ M86621: 1055-1078, 1517-1540. The specific forward and reverse primers used to identify the _1A-b splice variant were 5'-CTCAACACCATCGTGCTAATGATG-3' and 5'-CAGGTTGATGAAGTTATTCGGATT-3', respectively. We used the same sense and two different antisense oligonucleotides to examine whether the three known _1D subunit C-terminal splice variants (28) are expressed by GH₃ cells (Table I). Primers specific to exons of the rat _1D subunit gene are also listed in Table I. Amplification was carried out at 35 cycles with Pfu polymerase (Stratagene, La Jolla, CA). Each cycle was comprised of 30 s at 95 °C, 1 min at 55 °C, and 2 min at 72 °C. For the last cycle, annealing and extension times were increased to 3 and 10 min, respectively. Long-range RT-PCR amplification was carried out at 40 cycles with a Taq/Pfu polymerase mix (Stratagene, La Jolla, CA). The extension cycles were increased to 9 min at 72 °C, and the final extension time was 15 min. Q solution (Qiagen, Valencia, CA) was added to the PCR mix to obtain a maximal yield of product. For exon-specific PCR, individual clones of plasmid DNA were used as template, and amplification was carried out at 20 cycles with Taq polymerase (Stratagene). Each cycle consisted of 30 s at 95 °C, 60 s at 55 °C, and 45 s at 72 °C.

Determination of Relative Abundance of Alternatively Spliced _1D-Subunit Transcripts-- We used either RT-PCR or a hybridization strategy to examine the number and relative abundance of _1D subunit splice variants encoded by full-length _1D transcripts in GH₃ cells and rat brain. By using an oligonucleotide primer set designed to the 5'- and 3'-untranslated regions, respectively (Table I), full-length _1D-S transcripts encoding subunits with the short C terminus were amplified by RT-PCR generating a 5.1-kb product. For the full-length transcripts encoding _1D subunits with the long C terminus (_1D-L) we used the same sense primer to the 5'-untranslated region and an antisense oligonucleotide primer directed to the coding region 3' to the C-terminal splice locus, generating a 5.2-kb product. Due to the existence of additional splice sites, amplification products represented transcripts encoding multiple _1D-S and _1D-L isoforms. Subcloning PCR products en masse provided a means by which to determine the abundance of each particular variant. Upon transformation into an appropriate bacterial host, individual clones represented single isoforms and analysis of each provided the splice variation of the two upstream regions of known splicing activity. A PCR-based strategy was employed to analyze individual clones by designing exon-specific primers for each exon within the sites of splicing activity (Table I). The PCR products for each clone were visualized by gel electrophoresis and representative clones for each isoform were confirmed by sequencing. Therefore, by analyzing 100 individual clones, a percent abundance for each isoform could be obtained. Alternatively, we used dot blots of individual splice variant clones and tested the presence or absence of individual exons by using -³²P-labeled exon-specific primers. Oligonucleotides were end-labeled with [-³²P]dATP using T4 polynucleotide kinase (Ambion, Austin, TX), and 1.0 × 10⁶ dpm were added to the hybridization solution. Hybridization was carried out at 55 °C in 1 M NaCl, 50 mM Tris (pH 7.5), 5× Denhardt's, 0.1% sodium pyrophosphate, 0.5% SDS, and 10% polyethylene glycol 8000 for a minimum of 4 h. The blots were washed three times in 2× SSPE, 0.5% SDS at room temperature and autoradiography was carried out at 80 °C.

Creating Clones of _1D Subunit Splice Variants-- The long-range PCR products were digested by EcoRI generating multiple fragments with sizes of ~3.3 kb. These fragments were ligated into pBluescript SK() (Stratagene, La Jolla, CA) to generate pBlue Q.PCR_(x) (where x represents a specific splice variant). A rat _1D subunit clone (GenBank^TM accession number D38101), kindly provided by Dr. S. Seino (Chiba University, Chiba, Japan), digested partially with BamHI and fully with XhoI, was subcloned into pcDNA3.1+ (Invitrogen, Carlsbad, CA) generating C1.0. To generate a nucleotide sequence encoding the _1D-S C terminus, RT-PCR was performed on GH₃ cell RNA using amplimers flanking DraIII and XhoI sites. The DraIII/XhoI-cut PCR product was ligated into C1.0, generating Q1.0. Both Q1.0 and C1.0 were digested with EcoRI, gel-purified, and re-ligated to remove the 3.3-kb EcoRI fragment, generating Q1.1 and C1.1. The constructs _1D-S(x) and _1D-L(x) were generated by ligating the EcoRI fragment of pBlue Q.PCR_(x) into Q1.1 and C1.1, respectively. Sequencing revealed that cDNAs generated by long-range PCR often contained nucleotide sequence errors that would introduce incorrect amino acids into recombinant channels. Full-length _1D cDNAs were constructed from clones that had minimal sequence errors. Initial electrophysiological recordings from recombinant _1D-S channels made use of these clones. Subsequently, experiments examining current-voltage relationships, facilitation, and G protein coupling were repeated (n  3) demonstrating similar functional properties for sequence error-free recombinant _1D-S subunits. A construct that encoded an error-free _1D-S(31b) subunit was generated by ligating its 2-kb NotI/AgeI fragment to the 8-kb NotI/AgeI fragment of an error-free _1D-S clone identical in sequence 3' to the AgeI restriction site. The 3.3-kb EcoRI fragment of the _1D-S(31b) was then ligated to C1.1 to generate _1D-L(31b). The 3.2-kb BamHI fragment of _1D-S(31b) was ligated to a 6.8-kb BamHI fragment of an _1D-S(31a) clone to generate a construct that encoded an error-free _1D-S(31a) subunit.

DNA Sequencing-- Appropriate length PCR products were excised from ethidium bromide-stained gels and purified with QIAEX beads (Qiagen, Valencia, CA) according to the manufacturer's instructions. Purified DNA was subcloned into Bluescript SK() by blunt-end ligation and sequenced manually by ³²P-labeled dideoxynucleotide incorporation (Sequenase II, Amersham Pharmacia Biotech) and polyacrylamide gel electrophoresis. We used PCR-based cycle sequencing (FS chemistry; PE Biosystems, Foster City, CA) with custom internal primers (Life Technologies, Inc., Gaithersburg, MD) to sequence the ~3.3-kb PCR products subcloned into pBlue Q (i.e. pBlue Q.PCR_(x)). Sequences were obtained using an ABI Prism 377 genetic DNA analyzer, and data were analyzed using ABI Prism DNA sequencing software (PE Biosystems).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Opioid Receptors Couple to -Agatoxin IVa-resistant Ca²⁺ Channels in GH₃MORDOR Cells-- Ca²⁺ enters GH₃ cells through dihydropyridine-sensitive channels that can be inhibited by the activation of pertussis toxin-sensitive G proteins (24, 25, 29). Taken together with similar studies in pancreatic cell lines and neurons, these observations indicate the existence of G protein-sensitive L-type Ca²⁺ channels (21). However, because G_i/G_o proteins inhibit P/Q- and N-type channels (18), we re-examined the possibility that GH₃ cells may express either of these Ca²⁺ channel subtypes (Figs. 1 and 2). The P/Q-type channel antagonist -agatoxin IVa (10 nM) had no discernible effect on Ba²⁺ currents activated by depolarizing GH₃ cells stably expressing µ and  opioid receptors (GH₃MORDOR cells) (Fig. 1A). We used RT-PCR to examine whether GH₃ cells express transcripts encoding the _1A subunit, the principle component of P/Q-type channels (Fig. 1B). Transcripts encoding _1A subunits can be alternatively spliced giving rise to _1A-a and _1A-b subunits that may represent P- and Q-type Ca²⁺ channels, respectively (6). Low concentrations of -agatoxin IVa inhibit recombinant Ca²⁺ channels containing _1A-a subunits, whereas higher concentrations are required to inhibit channels formed by _1A-b subunits. We identified mRNA encoding the _1A-b subunit in GH₃ cells using RT-PCR analysis with a primer (see "Experimental Procedures") selective for this splice variant (Fig. 1B). In view of the presence of mRNA encoding the _1A-b subunit, we examined a potential role for -agatoxin IVa-sensitive channels in G protein-mediated inhibition of Ca²⁺ channel activity in GH₃MORDOR cells. Despite an inhibition of Ca²⁺ channel activity induced by -agatoxin IVa (500 nM), the  opioid receptor-selective agonist DPDPE (100 nM) continued to inhibit Ba²⁺ currents. Therefore, opioid receptors couple to -agatoxin-resistant Ca²⁺ channels in GH₃MORDOR cells (Fig. 1C). In later experiments we examined the properties of recombinant _1A channels providing further evidence for a lack of P/Q-type channels in GH₃ cells (Fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   -Agatoxin IVa-insensitive Ca2+ channels are inhibited by DPDPE. A, application of -agatoxin IVa (10 nM) does not significantly inhibit the Ba2+ current in GH3MORDOR cells. B, RT-PCR using a specific primer (Table I) demonstrates the presence of the 1A-b transcript. C and D, the  opioid receptor agonist DPDPE (100 nM) inhibited Ba2+ currents evoked by depolarizing GH3MORDOR cells from 80 to 0 mV (every 10 s). The inhibition was unaffected by the application of -agatoxin IVa (500 nM) at a concentration sufficient to block 1A-b Ca2+ channels (n = 3).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   GH3 cells have multiple Ca2+ channel subunit mRNAs. RT-PCR analysis of transcripts encoding 1B, 1C, 1D, 1S, 1, 2, 3, 4, and 2- subunits revealed multiple Ca2+ channel mRNAs in GH3 cells. Appropriately sized PCR products (arrows) were sequenced to confirm the existence of specific Ca2+ channel transcripts. Rat brain (RB) cDNA was used as a positive control for each subunit; additionally, rat skeletal muscle (RSM) cDNA was used as positive control for 1S.

Having ruled out a role for P/Q Ca²⁺ channels in the G protein modulation of Ba²⁺ currents recorded from GH₃MORDOR cells, we turned our attention to N-type channels. Electrophysiological studies demonstrate that GH₃ cells lack functional N-type Ca²⁺ channels (24). Furthermore, Lievano and colleagues (30) were unable to detect the _1B transcript in these cells using the ribonuclease protection assay. We confirmed a lack of mRNA encoding the _1B subunit in these cells using RT-PCR (Fig. 2).

GH₃ Cells Contain Multiple ₁ subunit Transcripts-- We synthesized cDNAs from RNA extracted from GH₃ cells, whole rat brain and rat skeletal muscle, using PCR to determine the complement of transcripts encoding Ca²⁺ channel ₁ subunits and accessory  and ₂- subunits (Fig. 2). We did not examine whether GH₃ cells express transcripts encoding T-type Ca²⁺ channels, because several studies have demonstrated their resistance to G protein modulation (31, 32). Furthermore, Ba²⁺ currents recorded from GH₃ cells lack a consistent T-type channel contribution (24). Specific forward and reverse oligonucleotide primers were designed for five cloned high voltage-activated ₁ subunits (_1B-1E, _1S),  subunits (_1-4), and an ₂- subunit (see "Experimental Procedures"). Sequencing of the bands excised from gels positively identified mRNAs encoding _1C and _1D subunits (confirming a previous report (30)) and _1-4 and ₂- subunits in GH₃ cells (Fig. 2). Because _1B, _1E, or _1S transcripts were absent and _1A channels do not contribute to the effects of DPDPE (Fig. 1), G protein-coupled inhibition of Ca²⁺ channels in GH₃ cells appears to occur through regulation of _1C and/or _1D channel activity. We considered the _1C subunit to be an unlikely candidate because of its inability to couple to opioid receptors (26). Therefore, we focused our attention on the _1D subunit believing this to be the most likely candidate for the G protein-sensitive L-type Ca²⁺ channel expressed by GH₃ cells.

Multiple _1D Splice Variants in GH₃ Cells-- Multiple splice variants of the _1D Ca²⁺ channel subunits exist in various cell types (7, 28, 33). In rat tissue, splicing causes variations in amino acid sequences located at three distinct loci: 1) between domains I and II, 2) between the S2 and S4 regions of domain IV, and 3) in the C-terminal region (Fig. 3). We used RT-PCR with oligonucleotide primer sets (Table I) spanning each of the three regions to investigate the presence or absence of splice variants in GH₃ cells (see "Experimental Procedures"). Gel extraction of candidate bands and subsequent sequencing confirmed multiple splice variants at all three splicing loci, resulting in numerous combinatorial _1D subtype possibilities (Fig. 3). Interestingly, analysis of the amino acid sequences in the C termini of _1D subunits indicated the presence of a truncated isoform containing the Gln-X-X-Glu-Arg (QXXER) motif, which has been observed previously in rat brain (33). We refer to _1D subunit variants that contain this truncated C terminus as _1D-S subunits. The QXXER motif is absent from the longer alternatively spliced _1D-L isoforms (Fig. 3). The fact that the QXXER sequence is present in _1A, _1B, and _1E subunits, which can all couple to inhibitory G proteins (15, 16), led to our hypothesis that this motif is sufficient to confer G protein sensitivity to recombinant channels formed by expressing cDNAs encoding _1D-S subunit variants.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Summary of the 1D subunit splice variants detected in GH3 cells. Three separate coding regions of the 1D Ca2+ channel subunit were investigated for the presence of splice variants by RT-PCR. Exons 10-13 correspond to the intracellular loop between domain I and II. Exons 30-33 correspond to a region between S1 and S5 of domain IV. Exons 40-49 correspond to the cytoplasmic tail. These three regions are indicated by a, b, and c, respectively. The identity of each PCR product was confirmed by sequencing. *, this splice fragment (c2) contains the putative G protein subunit-binding motif, whereas the other fragment, c1, does not.

Relative Abundance of Alternatively Spliced _1D Isoforms-- We used RT-PCR with exon-specific primers (Table I) and a hybridization strategy to examine the number of _1D subunit mRNA variants caused by the combinatorial possibilities enabled by virtue of the existence of the three known splicing hotspots depicted in Fig. 3 (see "Experimental Procedures"). This process was performed first using RNA isolated from GH₃ cells in which _1D-S and _1D-L variants were examined and subsequently, for _1D-S variants specifically, using rat whole brain RNA (Fig. 5). Bands containing a mixture of all cDNAs encoding _1D-S or _1D-L subunits were excised from agarose gels (Fig. 4A). We used EcoRI to excise cDNA sequences encoding the two initial regions of splicing activity. These were then ligated into pBluescript allowing transformation of bacteria. We picked 100 colonies of bacteria transformed with the vector containing EcoRI fragments of either _1D-S from GH₃ cells or rat brain, or _1D-L from GH₃ cells. We then employed PCR or exon-specific hybridization, with specific oligonucleotides (Table I) to identify exons introduced by alternative splicing into cDNAs isolated from individual bacterial colonies (Fig. 4B).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Isolation and identification of 1D transcripts expressed by GH3 cells and rat brain. A, amplification of full-length 1D-S transcripts containing the putative G binding domain (denoted by +QXXER) and partial-length 1D-L transcripts (denoted by QXXER). B, PCR with exon-specific amplimers was used to analyze 1D-S clones in GH3 cells and rat brain. Numbers above each lane denote exons recognized by each primer set. Shown are representatives from a GH3 cell (top) and a rat brain (bottom) 1D-S clone.

Expression of Recombinant 1D Subunits-- We made three full-length cDNAs encoding _1D-S(31a), _1D-S(31b), and _1D-L(31b) subunits. The _1D-S(31a) and _1D-S(31b) cDNAs encode the most abundant _1D-S subunit variants detected in the GH₃ cells and rat brain, respectively (Fig. 5). These subunits differ in the variety of alternatively spliced exon 31 (variants 3 and 4, respectively, in Table II). The _1D-L(31b) subunit sequence differs from the _1D-S(31b) subunit sequence at the C terminus (see Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Percent abundance of 1D isoforms in rat brain and GH3 cells. A total of 100 individual clones for rat brain 1D-S (bars with lines), GH3 cell 1D-S (checkered bars), and 1D-L (solid bars) isoforms were analyzed by PCR or hybridization to determine the percent abundance of each splice variant. The 1D-S variants 3 (1D-S(31a)) and 4 (1D-S(31b)) are the most prevalent in GH3 cells and brain, respectively. The numbering scheme for 1D subunit splice variants is provided in Table II.

View this table: [in this window] [in a new window]   Table II Summary of 1D-S variants in GH3 cells PCR with exon-specific primers revealed multiple variants terminating with exon 41a, i.e. 1D-S. Numbers in the column represent the nomenclature assigned to each type of variant. Exons are shown in the top row.

We examined the functional properties of the _1D-S(31a), _1D-S(31b), and _1D-L(31b) subunit variants by transiently introducing their cDNAs with accompanying _2a and ₂- cDNAs into HEK or CHO cells either in the absence or presence of stably expressed  opioid receptors (HEKDOR and CHODOR cells, respectively). The properties of the expressed channels were compared with recombinant channels formed by the expression of cDNAs encoding _1A, _2a, and ₂- subunits (Fig. 6). Currents were recorded using the whole-cell patch-clamp technique with Ba²⁺ as the charge carrier (see "Experimental Procedures"). The voltage dependence of current activation was investigated by depolarizing cells from 80 mV to between 50 and 60 mV (10-mV increments) for 80 ms. Ba²⁺ currents had a threshold of activation of around 45 mV and peaked at 0 mV for _1D-S(31a), _1D-S(31b), and _1D-L(31b) channels (Fig. 6B). These values were similar to those reported for Ba²⁺ currents recorded from GH₃ cells under the same conditions (22). By contrast, currents recorded from cells expressing _1A subunits had significantly higher voltages at which threshold of activation and peak current amplitudes occurred (~30 and 10 mV, respectively, Fig. 6B). There was no significant differences between the peak current densities for the _1D-S(31a), _1D-S(31b), and _1D-L(31b) channels, which were 75 ± 23 picoamps (pA)/picofarads (pF) (n = 5), 71 ± 12 pA/pF (n = 5), and 49 ± 5 pA/pF (n = 3), respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Current-voltage relationship of recombinant 1D Ca2+ channels. Cells were transiently transfected with cDNAs encoding 1D, 2a, and 2- subunits. A, superimposed currents activated by depolarizing cells expressing 1D-S(31b) subunits to between 50 and 60 mV from 80 mV. B, mean normalized I-V curves for 1A (, n = 9), 1D-S(31b) (, n = 6), 1D-S(31a) (, n = 4), and 1D-L(31b) channels (, n = 6). Currents were normalized to peak amplitude 15 ms after the onset of test pulse. C, a CHO cell expressing 1D-S(31b) subunits was depolarized from 100 mV to 30, 10, and 50 mV in the top, middle, and bottom traces, respectively. A single-exponential fit, shown superimposed on each current trace, was used to determine the time constant of deactivation ( = 0.2 ms) and tail current amplitude. D, a plot of the tail current amplitude (expressed as percent maximum tail current) against voltage was generated for 1D-S(31a) () and 1D-S(31b) () subunits. Data points were fitted using the Boltzmann equation (see "Experimental Procedures").

A failure of Ba²⁺ currents mediated by _1D channels to reverse (presumably due to low Cs⁺ permeability) prevented accurate determinations of voltages for half-maximal activation by fitting the current-voltage relationships in Fig. 6B. Therefore, we performed tail current analysis to determine the voltage required for half-maximal channel activation (V_1/2) for currents mediated by _1D-S(31a) and _1D-S(31b) subunits. Tail currents were well fitted by single exponentials with time constants of 0.2 ms in both cases (Fig. 6C). The relationships between tail current amplitude and voltage for _1D-S(31a) and _1D-S(31b) subunits were similar. Fitting the data points with a Boltzmann function (see "Experimental Procedures") yielded V_1/2 values of 2.9 and 3.9 mV for _1D-S(31a) and _1D-S(31b) subunits, respectively (Fig. 6D).

The dihydropyridine sensitivity of Ba²⁺ currents recorded from cells expressing the _1D-S(31b) subunit was similar to that of Ba²⁺ currents recorded from GH₃ cells (24), whereas currents recorded from cells expressing the _1A subunit were significantly less sensitive to nimodipine (Fig. 7). A previous study demonstrated that nimodipine caused a more potent inhibition of Ca²⁺ channels when GH₃ cells were depolarized from a holding potential of 40 mV rather than one of 80 mV (24). Likewise, the nimodipine inhibition of currents mediated by _1D-S(31b) subunits was potent in cells held at 40 mV (IC₅₀ = 0.25 ± 0.02 µM). Nimodipine inhibited Ba²⁺ currents recorded from cells held at 80 mV with an IC₅₀ of 1.0 ± 0.2 µM. Recombinant _1A channels were inhibited by nimodipine with an IC₅₀ of 49 ± 1 µM in cells voltage-clamped at 40 mV. Taken together, these data support our assertion that, despite the presence of detectable levels of mRNA encoding the _1A subunit in GH₃ cells (Fig. 1), their functional channels are predominantly L-type.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Nimodipine sensitivity of 1A and 1D channels. Concentration-response relationship for the inhibition of 1A (, n = 6) and 1D-S(31b) (, n = 5) Ca2+ channels by nimodipine. Cells were held at 40 mV, and currents were recorded in the presence of 30 mM Ba2+ to minimize voltage-dependent inactivation. The concentration-response relationship of 1D-S(31b) Ca2+ channels for inhibition by nimodipine was shifted to the right when cells were held at 80 mV (, n = 5). Curves were generated by a logistic equation (see "Experimental Procedures").

G Protein Sensitivity of Recombinant Ca²⁺ Channels-- Cloned µ and  opioid receptors couple to L-type Ca²⁺ channels in GH₃MORDOR cells through activation of inhibitory G_i/o proteins (25). We hypothesized that the G protein-sensitive Ca²⁺ channel component is comprised of an _1D subunit. Furthermore, we anticipated that the QXXER motif within specific _1D-S subunits was necessary for binding and therefore channel inhibition. Here we examined whether the QXXER motif is sufficient to confer opioid receptor coupling or G protein sensitivity to recombinant channels formed by the _1D-S subunit coexpressed in HEKDOR cells with _2a and ₂- subunits.

The  opioid receptor-selective agonist DPDPE (100 nM) caused a voltage-dependent inhibition of _1A channel activity recorded from transiently transfected HEKDOR cells (Fig. 8A). By contrast,  receptor activation had no effect on the activity of either _1D-S(31b) or _1D-L(31b) channels expressed in HEKDOR cells demonstrating that the QXXER motif is not sufficient to confer opioid receptor coupling (Fig. 8B). We transiently transfected cDNAs encoding the Ca²⁺ channel subunits into CHO cells stably expressing  receptors (CHODOR cells) to examine whether a different expression environment may enable coupling. Once again, DPDPE (100 nM) inhibited _1A channel activity but had no effect on the activity of either of the two _1D splice variants tested (in each case n = 4). We investigated whether direct activation of G proteins would cause a voltage-dependent inhibition of recombinant channels formed by _1D-S subunits. Depolarization of CHO cells expressing either _1D-S(31b) or _1D-L(31b) subunits with GTPS (300 µM) in the electrode solution did not significantly alter the level of Ba²⁺ current facilitation seen with the double-pulse protocol in the absence of GTPS (F_max = 34.8 ± 1.7%, F₅₀ = 5.2 ± 3.1 mV versus F_max = 30.8 ± 2.3%, F₅₀ = 0.9 ± 4.3 mV and F_max = 35.8 ± 0.79%, F₅₀ = 11.2 ± 1.3 mV versus F_max = 24.5 ± 3.92%, F₅₀ = 19.9 ± 1.93 mV, respectively, Fig. 9A and B). We repeated these experiments by expressing _1D-S(31a) subunits in CHO cells to determine whether G protein coupling occurs in the exon 31 variant that predominates in GH₃ cells. GTPS had no significant effect on basal Ba²⁺ current facilitation compared with control (F_max = 33.7 ± 2.7%, F₅₀ = 1.6 ± 4.4 mV versus F_max = 24.5 ± 1.3%, F₅₀ = 7.9 ± 3.0 mV, Fig. 9C). Similar experiments also indicated a lack of G protein coupling when _1D-S(31b) or _1D-L(31b) subunits were expressed in HEKDOR cells (n = 6 and 4, respectively). Because opioid receptor coupling to Ca²⁺ channels is thought to be mediated by G_o (34), CHO, CHODOR, and HEKDOR cells were transiently transfected with G_oA or G_oB cDNAs to determine whether the lack of coupling is due to inadequate levels of these G proteins. Neither the overexpression of G_oA (n = 5 and 4 for HEKDOR and CHODOR, respectively) nor G_oB (n = 3 and 5 for HEKDOR and CHO) resulted in G protein-mediated inhibition of _1D-S(31b) channels by GTPS. Syntaxin-1A may play a permissive role in G-mediated inhibition of N-type Ca²⁺ channels (35). We examined whether this may also be the case for _1D-S channels. No Ba²⁺ current inhibition was observed in the presence of GTPS (n = 3) in CHO cells transiently transfected with cDNAs encoding syntaxin-1A and _1D-S(31b), _2a, and ₂-. These data demonstrate that the QXXER motif is not sufficient to confer sensitivity of _1D-S(31a) or _1D-S(31b) subunits to activated G proteins.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Opioid-receptors couple to 1A channels but not to either of the 1D channel variants tested. A, top, Ba2+ currents through 1A channels before, during, and after application of the  opioid receptor agonist DPDPE. Bottom, voltage-dependent reversal of inhibition. Current amplitudes were normalized to the value obtained after a prepulse to 50 mV (I1) and plotted as a function of the pre-pulse potential. Mean data from five cells in the presence of DPDPE were fitted using the Boltzmann equation (see "Experimental Procedures"). The fit indicated that currents in the presence of DPDPE were maximally facilitated (Fmax) by 107%, and half-maximal facilitation (F50) occurred at 6.5 mV. B, top, 1D-S(31b) and bottom, 1D-L(31b) Ba2+ currents were not inhibited by DPDPE (100 nM).

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Effect of GTPS on 1D splice variants. The double-pulse recording protocol was used to determine whether 1D channels were inhibited in the presence of GTPS (300 µM). Data points are averages of at least five determinations. A-C, no significant difference in the level of 1D-S (31b) (), 1D-L (31b) (), and 1D-S (31a) () current facilitation was observed when GTP--S was included in the recording electrode (filled symbols).

Voltage-dependent Facilitation of _1D Channel Activity-- Depolarizing pre-pulses caused voltage-dependent facilitation of Ba²⁺ currents mediated by all three recombinant _1D channel variants (Fig. 9). We investigated the effect of pre-depolarization on the current-voltage relationship of _1D-S(31b) Ca²⁺ channels. An increase in peak current and a transient leftward shift in the current-voltage relationship occurred after application of a 60-mV depolarizing pre-pulse (I_peak = 938 pA versus I_peak = 1097 pA for pre- and post-pulse, respectively; Fig. 10A). Current amplitude was unaffected at voltages greater than 30 mV. We also examined the time required for recovery from voltage-dependent facilitation in cells expressing _1D-S(31b) channels (Fig. 10B). A single-exponential fit of the decline in current amplitude following a 60-mV pre-pulse yielded the time constant for recovery from facilitation ( = 58.6 ± 10.5 ms). The rapid time course of recovery indicates that this event is unlikely to involve protein phosphorylation. This time course is similar to those reported previously for voltage-dependent reversal of G protein inhibition of _1A and _1B channels (36). However, G protein activation through inclusion of GTPS in the recording electrode had no effect on the observed facilitation of _1D-S(31b) channels (Fig. 9A). We examined further whether the facilitation of _1D channel activity was caused by reversal of constitutive G protein coupling. The reversal of G_i/o protein-mediated inhibition of Ca²⁺ channel activity is known to depend on the concentration of activated G proteins (37). However, pretreatment of cells with pertussis toxin (100 ng/ml) 24 h prior to recording, a procedure that should significantly reduce G_i/o activation, had no significant effect on the time course for the recovery from facilitation ( = 51.5 ± 5.3 ms). Furthermore, transfection of cells with a cDNA encoding the -adrenergic receptor kinase (-ARK) minigene peptide thought to inhibit G from binding to its effectors (38) had no significant effect on the time constant of recovery from facilitation ( = 69.8 ± 19.5 ms). Our data therefore suggest that the observed voltage-dependent facilitation of _1D-S(31b) channels is independent of G protein activity.

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Voltage-dependent facilitation of 1D channels. A, voltage-dependent facilitation of currents was investigated by comparing the current-voltage relationship before and after a depolarization to 60 mV. Left, superimposed Ba2+ currents recorded from a CHO cell expressing 1D-S(31b). Right, graph showing the relationship between current amplitude and voltage before () and after () a depolarization to 60 mV. B, left, superimposed currents mediated by 1D-S(31b) channels during a prepulse to 60 mV and in response to a test pulse to 0 mV 10-140 ms following the prepulse. Right, a graph of the time required for recovery from voltage-dependent facilitation. The peak current amplitude was measured and expressed relative to the peak amplitude of the maximally facilitated current, i.e. the first current (I1) after the depolarizing pulse. Curves are exponential fits to the data. Neither pre-treatment with pertussis toxin (100 ng/ml, ) nor co-expression of the -ARK minigene () caused a significant change in the time required for recovery from voltage-dependent facilitation when compared with control (). Data points are averages of at least four determinations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we identified mRNAs encoding three ₁ (_1A, _1C, and _1D), four , and ₂- Ca²⁺ channel subunits in GH₃ cells. The transcripts encoding _1B and _1S subunits were not present. Despite detectable levels of _1A-b subunit mRNA, which would be expected to encode Q-type Ca²⁺ channels, Ba²⁺ currents recorded from GH₃MORDOR cells are almost abolished by the dihydropyridine nimodipine (24). Furthermore, -agatoxin IVa (500 nM) at a sufficient concentration to block _1A-b channels (6) did not affect the inhibition of Ba²⁺ currents induced by  opioid receptor activation. Together, these observations suggest that P/Q-type channels do not contribute to the opioid receptor-sensitive Ca²⁺ channel population in GH₃MORDOR cells. The highest concentration of -agatoxin IVa (500 nM) tested did cause an inhibition of Ba²⁺ current amplitude. There are two possible explanations for this observation. Functional Q-type Ca²⁺ channels that are unable to couple to opioid receptors may exist in GH₃MORDOR cells. Alternatively -agatoxin IVa (500 nM) may inhibit a subset of L-type Ca²⁺ channels (perhaps _1C) that do not couple to opioid receptors. Further experiments will be required to distinguish between these two possibilities. Taken together these data support the hypothesis that opioid receptors can couple to dihydropyridine-sensitive L-type Ca²⁺ channels in GH₃MORDOR cells. However, the fact that µ and  receptor activation inhibits Ca²⁺ channel activity in GH₃MORDOR cells by <20% on average suggests that not all of their L-type channels are G protein-regulated (25).

Both _1C and _1D subunits may participate in the L-type Ca²⁺ channel activity of GH₃ cells. However, recombinant µ receptors failed to couple to _1C subunits when both were expressed in Xenopus oocytes (26). Therefore, we hypothesized that _1D subunits couple to opioid receptors in GH₃MORDOR cells (25). RT-PCR analysis revealed three loci within the _1D gene at which alternative splicing occurs in GH₃ cells. Transcripts encoding _1D subunits are also alternatively spliced at these sites in human and rat brain, hamster insulin-secreting cells, and chicken cochlea (7, 33, 39, 40). The observation that alternative splicing occurs at the C terminus of the gene producing transcripts of differing lengths was of particular interest; the shorter transcript (_1D-S) (33) ends with a sequence that encodes an amino acid motif (QXXER) found in all other Ca²⁺ channel ₁ subunits (_1A, _1B, and _1E) that couple to G proteins. This motif is also present in other effectors, including adenylyl cyclase 2, G protein-activated inward rectifying K⁺ channels, and -adrenergic receptor kinase (-ARK), which all couple in a membrane-delimited fashion to inhibitory metabotropic receptors (16). Several lines of evidence support the hypothesis that subunits mediate the inhibitory actions of G protein-coupled receptors on Ca²⁺ channels, and inhibition is thought to occur subsequent to the binding of the subunit to the QXXER domain (15, 18, 37).

In view of their putative role in G protein signaling, we used long range RT-PCR to identify the alternatively spliced transcripts encoding _1D subunits found in GH₃ cells and rat whole brain. The relative abundances of _1D-S and _1D-L variants in GH₃ cells were determined by analyzing 100 transformed bacterial clones for each. Similarly, the number of _1D-S variants was examined in whole rat brain. In retrospect, the large number of isoforms detected was not surprising considering the presence of multiple hotspots of splice variation. However, the number of observed variants was larger in GH₃ cells than in brain. The additional splicing may be a characteristic of GH₃ cells. Alternatively, analyzing 100 clones in the whole brain might not be sufficient to detect the presence of specific variants, which may only occur in small subpopulations of neurons. Nonetheless, analysis of the transcripts in the whole brain confirmed that the majority of splicing activity was not exclusive to GH₃ cells.

To date, there have been relatively few studies demonstrating functional expression of _1D Ca²⁺ channel variants (41-44). Analysis of the biophysical properties of the three _1D variants expressed in our study revealed that they have a similar current-voltage relationship to native _1D currents of the cochlear inner hair cells, activation threshold occurred at around 45 mV, and currents peaked near 0 mV (45). By contrast, _1C channels expressed with _2a and ₂- subunits had a higher threshold of activation and peaked at 20 mV (46).

Voltage-dependent inhibition by dihydropyridine antagonists distinguishes L-type channels from all other Ca²⁺ channels (47). Nimodipine blocked _1D-S(31b) Ca²⁺ channels with an approximately 4-fold lower IC₅₀ at 40 mV holding potential than at 80 mV. The nimodipine sensitivity of Ba²⁺ currents through _1D-S(31b) channels was similar to that of native GH₃ cell Ca²⁺ channels (24). Intriguingly, Bell and colleagues (44) did not observe a voltage-dependent block of expressed _1D channels by dihydropyridine antagonists. This divergence may be due to the _1D isoforms used. We characterized the dihydropyridine sensitivity of the short isoform, _1D-S(31b), terminating with exon 41a. Bell and colleagues (44) employed the long isoform that also lacked exon 11 and contained exon 31a. However, this explanation seems unlikely, because the IIIS5, IIIS6, and IVS6 regions of the L-type Ca²⁺ channels, thought to be involved in dihydropyridine sensitivity (48), are the same in the two expressed _1D variants.

We chose the predominant _1D-S transcripts in GH₃ cells and rat brain, _1D-S(31a) and _1D-S(31b), respectively, to assess whether the QXXER motif is sufficient to confer G protein coupling to these channels. We compared their sensitivity to activated G proteins to that of _1D-L(31b) subunits that lack the QXXER motif. Activation of G proteins by either opioid receptors or GTPS failed to inhibit Ba²⁺ currents mediated by any of the _1D variants. Experiments in NG108-15 cells identified G_o as the inhibitory G protein required for the inhibition of Ca²⁺ channels by opioids (34). Functional analysis of _1D-S(31b) and _1D-L(31b) in CHODOR cells, whose expression of endogenous G_o is well established (49), revealed a lack of G protein-mediated inhibition of _1D Ca²⁺ channels. Moreover, increasing the levels of either G_oA or G_oB in HEKDOR and CHO cells by transient transfection with the appropriate cDNAs had no effect on the lack of G protein coupling to _1D channels.

Recently, Jarvis and colleagues (35) demonstrated a role for syntaxin-1A in the augmentation of G protein-mediated inhibition of _1A channels. Furthermore, syntaxin-1A was shown to colocalize in the plasma membrane and co-immunoprecipitated with _1D Ca²⁺ channels in pancreatic beta cells (50). We postulated that syntaxin-1A might be required for G protein-mediated modulation of _1D Ca²⁺ channels. However, transient expression of syntaxin-1A into CHO cells did not result in G protein-mediated inhibition of _1D channels.

All _1D splice variants examined exhibited a potentiation or facilitation evoked by depolarizing pre-pulses. Facilitation was independent of G protein activation, and the rapid rate of recovery ( < 70 ms) suggests that it is unlike the phosphorylation-dependent facilitation seen in recordings of L-type Ca²⁺ channel activity in bovine chromaffin cells where reversal of facilitation is slow (~60 s) (51). Immunohistochemical analysis of the distribution of _1D subunits in neurons reveals their distribution on cell bodies and proximal dendrites in the hippocampus and many other brain regions (52, 53), where they may participate in the activity-dependent initiation of gene expression that is inhibited by dihydropyridines (54, 55). Furthermore, their voltage-dependent facilitation may also enable activity-dependent enhancement of Ca²⁺ influx contributing to long-term potentiation and depression (56-58).

Our results suggest that the QXXER motif is not sufficient to confer G protein sensitivity to _1D channels. Recent reports suggest that, in addition to the two proposed G sites in the intracellular loop between domains I and II, domain I and N- and C-terminal regions of _1B, and _1E Ca²⁺ channels may be important for G protein coupling (36, 59, 60). Similarly, multiple regions may be required for G protein-mediated inhibition of _1D channels to occur. Thus other _1D splice variants may be responsible for the G protein-sensitive L-type Ca²⁺ channels in GH₃ cells. Alternatively, the required signaling molecules for G protein-mediated inhibition of L-type Ca²⁺ channels present in GH₃ cells may not be present in HEK or CHO cells.

There are few studies of functional _1D channels, and this is among the first to examine multiple alternatively spliced isoforms. We have shown that alternatively spliced _1D subunits form functional L-type channels that resemble those of GH₃ cells in their voltage dependence of activation and nimodipine sensitivity.

	ACKNOWLEDGEMENT

We thank Megan Dankovich for expert technical assistance with automated sequencing and tissue culture.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Medical Center, The George Washington University, 2300 Eye St. NW, Washington, DC 20037. Tel.: 202-994-3546; Fax: 202-994-2870; E-mail: phmtgh@gwumc.edu.

Published, JBC Papers in Press, August 20, 2001, DOI 10.1074/jbc.M103724200

	ABBREVIATIONS

The abbreviations used are: RT-PCR, reverse transcription-polymerase chain reaction; HEK, human embryonic kidney; GTPS, guanosine 5'-3-O-(thio)triphosphate; CHO, Chinese hamster ovary; DPDPE, [D-Pen²,D-Pen⁵]enkephalin; kb, kilobase(s); -ARK, -adrenergic receptor kinase; DOR, opioid receptor; MOR, µ opioid receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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