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J. Biol. Chem., Vol. 279, Issue 6, 4102-4109, February 6, 2004

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Identification of Sites Responsible for Potentiation of Type 2.3 Calcium Currents by Acetyl--methylcholine^*

Ganesan L. Kamatchi, Ruthie Franke, Carl Lynch, III, and Julianne J. Sando

From the Department of Anesthesiology, University of Virginia Health Sciences Systems, Charlottesville, Virginia 22908-0710

Received for publication, August 5, 2003 , and in revised form, November 14, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To address mechanisms for the differential sensitivity of voltage-gated Ca²⁺ channels (Ca_v) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M₁ receptors and different Ca_v 1 subunits, all with 1B,2/ subunits. Acetyl--methylcholine (MCh) decreased Ca_v 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Ca_v 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Ca_v 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the ₁ subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the ₁ 2.3 subunits that respond to MCh. Quadruple Ser Ala mutation of the predicted MCh sites in the ₁ 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II–III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser Ala mutation of just the II–III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II–III linker of ₁ 2.3 subunits mediate the responsiveness of Ca_v 2.3 channels to MCh. Because Ca_v 2.3 channels contribute to action potential-induced Ca²⁺ influx, these sites may account for M₁ receptor-mediated regulation of neurotransmission at some synapses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Voltage-gated Ca²⁺ (Ca_v)¹ channels comprise a large family of heteromultimeric proteins containing a pore-forming ₁ subunit and auxiliary , 2/, and subunits. Electrophysiological and pharmacological characterizations have determined that ₁ 1.1, 1.2, 1.3, and 1.4 subunits encode L-type channels; ₁ 2.1 encodes P/Q-type channels; ₁ 2.2 encodes N-type channels; ₁ 2.3 encodes R-type channels; and ₁ 3.1, 3.2, and 3.3 encode T-type Ca_v channels (1, 2). The ₁ subunit consists of four domains (I–IV), each with six transmembrane segments and is modulated by the binding of auxiliary 2/ and subunits (3). The intracellular segments of the ₁ subunit, namely the N and C termini and the intracellular loops between domains I and II, II and III, and III and IV, possess the binding/recognition sites for second messengers such as G protein subunits or intracellular Ca²⁺ ([Ca²⁺]_i) as well as sites that can be phosphorylated by protein kinase C (PKC) (4–7).

Members of the Ca_v family are variably influenced by agents that activate PKC. For example, Ca_v 1.2a (rabbit heart) currents expressed in Xenopus oocytes were potentiated by the PKC activator phorbol 12-myristate 13-acetate (PMA) (8, 9); however, the same channel when expressed in tsA-201 cells, a subclone of the human embryonic kidney cell line HEK-293, was inhibited by PMA (10). In contrast, Ca_v 1.2c (rat brain and human heart) currents expressed in Xenopus oocytes were not influenced by PMA (11, 12). Among the members of the Ca_v 2.0 family, Ca_v 2.1 currents were not affected by PMA, whereas Ca_v 2.2 and 2.3 currents were potentiated (4–6, 13). Presumably, phosphorylation of certain amino acids alters the gating of channels, leading to greater currents. The sensitivity of Ca_v channels to PMA suggests that some of these channels may represent potential targets for certain hormones, neurotransmitters, and agonists that activate PKC by a receptor-mediated pathway. For example, stimulation of the odd numbered (M₁, M₃, and M₅) muscarinic receptors results in the activation of PKC (14). Differential results were obtained when Xenopus oocytes coexpressing M₁ receptors and Ca_v 1.2c or Ca_v 2.3 channels were exposed to MCh. Ca_v 1.2c currents were decreased by MCh (15), but Ca_v 2.3 currents were potentiated, possibly due to M₁ receptor-induced activation of PKC (13).

PKC isozymes can be divided into three categories: classic PKCs or cPKCs, including PKC , I, II, and isozymes that require Ca and are stimulated by phosphatidylserine and diacylglycerol (DAG); novel or nPKCs (, , , and ), which are Ca-independent but still stimulated by phosphatidylserine and DAG; and atypical or aPKCs (, /), which are Ca- and DAG-independent. In a recent study, we suggested that cPKCs may be responsible for the action of MCh, whereas nPKCs may contribute to the action of PMA (13). Unique cPKC- and nPKC-selective phosphorylation sites may be present in the ₁ 2.3 subunit.

Here we have compared currents among Ca_v 1.2c, 2.1, 2.2, and 2.3 channels expressed in Xenopus oocytes also expressing M₁ receptors. Ca_v 1.2c currents were decreased by MCh (15) but unaffected by PMA. Neither MCh nor PMA affected 2.1 currents. Ca_v 2.2 and 2.3 currents were potentiated by PMA, but MCh increased only 2.3 currents, as shown before (13, 16). Based on the comparison of amino acid sequences in the channel types, we have mutated unique potential serine/threonine phosphorylation sites in the ₁ 2.3 subunit to alanine and coexpressed the mutants with M₁ receptors in Xenopus oocytes. Specific MCh-sensitive sites have, thus, been identified.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of Mutants—Selected serines or threonines were mutated to alanines by using overlap extension PCR mutagenesis with Pfu Turbo DNA polymerase (QuikChange site-directed mutagenesis method, Stratagene, La Jolla, CA). Oligonucleotide primers containing the desired mutations were used to extend the template, the wild type rat brain ₁ 2.3 subunit cDNA subcloned in the pMT2 vector. The PCR product was treated with DpnI, a restriction enzyme specific for methylated and hemimethylated DNA to digest the template DNA. The digested PCR product was transformed in Escherichia coli using the protocol from the supplier, and the DNA from the selected transformants was processed for sequencing. The whole coding region of the construct was sequenced (Biomolecular Research Facility, University of Virginia) to confirm the planned mutation and the absence of unwarranted mutations contributed by the PCR reaction.

Harvesting of Oocytes and cDNA Injection—Mature female Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI), housed in an established frog colony, and fed regular frog brittle on alternate days. For the removal of oocytes, a frog was anesthetized in 500 ml of 0.2% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) in water until unresponsive to a painful stimulus. The anesthetized frog was placed supine on ice, and an incision of 1.5 cm in length was made through both the skin and muscle layers of one lower abdominal quadrant. A section of the ovary was exteriorized and a lobule of oocytes (500) was removed. The wound was closed in two layers, and the animal was allowed to recover from anesthesia, kept in a separate tank overnight, and returned to the colony the following day. The oocytes were washed twice in calcium-free OR2 solution (in millimolar: NaCl 82.5, KCl 2, MgCl₂ 1.8, HEPES 5, pH 7.5) and transferred to OR2 solution containing 1 mg/ml collagenase (type 1A, Sigma). The dish containing the oocytes in collagenase solution was agitated for a period of 2–3 h at room temperature to remove the follicular cell layer. Defolliculation was confirmed by microscopic examination. Following this, the oocytes were washed in OR2 solution and transferred to modified Barth's solution (in millimolar: NaCl 88, KCl 1, NaHCO₃ 2.4, CaCl₂ 0.41, MgSO₄ 0.82, HEPES 15, pH 7.4) containing 2.5 mM sodium pyruvate and 10 µg/ml gentamicin sulfate. The oocytes were allowed to recover by incubation at 16 °C for 3–10 h before cDNA injection. Nuclear (germinal vesicle) injection was performed (Drummond "Nanoject," Drummond Scientific Co., Broomall, PA) using a maximum of 4 ng of cDNA containing 3 ng of a 1:1:1 mix (molar ratio) of Ca_{v 1}, 1B, 2/ cDNA subunits and 1 ng of rat M₁ receptor cDNA in pcDNA 3.1 (Invitrogen, Carlsbad, CA). The oocytes were returned to Barth's solution and incubated at 16 °C for 6–8 days before the recording of current.

Current Recording—Macroscopic currents, with Ba²⁺ (I_Ba) as the charge carrier, were recorded employing a two-electrode voltage-clamp technique using Oocyte Clamp OC-725C (Warner Instrument Corp., Hamden, CT). The amplifier was linked to an interface and an IBM-PC-compatible computer equipped with pClamp software (version 8.2, Axon Instruments, Foster City, CA) for data acquisition and analysis. Leak currents were subtracted using the P/4 procedure. Microelectrodes with an agarose cushion were filled with 3 M CsCl; typical resistances were 0.5–2.5 M. KCl-Agar bridges were used as ground electrodes to minimize any junction potential attributable to changes in ionic composition of the bath solution. The oocytes were placed in a recording chamber (500-µl volume) superfused with the recording solution containing (in millimolar): Ba(OH)₂ 40, NaOH 50, KOH 2, HEPES 5, using methanesulfonate as the anion to adjust the pH to 7.4. Niflumic acid (0.4 mM) was included to block intrinsic Cl channels. Oocytes were held at –80 mV before being depolarized to the test potential. A test potential of 0 mV for a duration of 850 ms was employed in oocytes expressing any Ca_v channel except Ca_v 2.2 channels. The same protocol was used for the depolarization of Ca_v 2.2 channels, except that the test potential was 20 mV. The current-voltage (I-V) relationship of oocytes expressing the wild type Ca_v 2.3 channel or a quadruple mutant were determined. The I-V was recorded for a duration of 450 ms using step depolarizations from –50 mV to 100 mV in 10-mV increments.

Drug Treatment—All of the oocytes exhibiting I_Ba greater than 400 nA underwent control, treatment, and wash protocols. The control I_Ba was recorded at the 8th min after the oocyte was impaled. MCh was used to activate M₁ receptors. Wherever necessary, the effect of PMA also was tested in oocytes coexpressing M₁ receptors. MCh or PMA was perfused for 60 s, and the current was recorded after another 60 s, thus exposing the oocyte to the agonist for a period of 2 min.

Chemicals—PMA (Calbiochem, San Diego, CA) and MCh (Sigma) were dissolved in Me₂SO (0.1%) and distilled water, respectively. MCh and PMA were prepared as concentrated stock solutions and stored frozen at –20 °C. They were diluted to their final concentration in recording solution on the day of the experiment. To block endogenous Cl^– currents, niflumic acid (Sigma) was added to the recording solution, which was stirred overnight for it to dissolve.

Data Analysis—The data are shown as means ± S.E., unless otherwise indicated. The peak represented the maximum amplitude of the inward current. The current amplitude at 830 ms (of the total period of 850 ms) was arbitrarily defined as the late current, which was used as a measure of relative degree of channel inactivation. The current inactivation rate was fit by a single exponential function using the equation,

(Eq. 1)

where A is the coefficient of the exponential, t is time (ms), is the time constant (ms), and C is the fraction of residual current. Statistical significance was determined using paired or unpaired t test and p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
MCh or PMA Differentially Affect Various Ca_v Channels Expressed in Xenopus Oocytes—Currents in oocytes expressing various Ca_v channels were examined with either 1 µM MCh, the EC₅₀ for MCh observed in our recent study (13), or PMA (100 nM). PMA failed to affect Ca_v 1.2c currents (percent change: peak current = –1.1 ± 1.5; late current –2.6 ± 1.2; mean ± S.E., n = 4) significantly. Similarly Ca_v 2.1 currents (percent change: peak current = 0.9 ± 0.5; late current 1.9 ± 5.8; mean ± S.E., n = 5) were also unaffected by PMA. Under the same conditions, PMA potentiated Ca_v 2.2 (percent change: peak current = 37.2 ± 5.4; late current 99.9 ± 28.9; mean ± S.E., n = 7) (Fig. 1) and Ca_v 2.3 currents (see Figs. 1 and 4) significantly.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Effect of PMA (100 nM) on Cav currents coexpressed with 1, 1B, 2/ subunits and muscarinic M1 receptors in Xenopus oocytes. The oocytes injected with the cDNA were voltage clamped after 7–8 days of incubation at 16 °C. They were held at –80 mV and depolarized to the test potential of 0 mV for all types of Cav channels except Cav 2.2, in which a test potential of 20 mV was used. The drug treatment and the current recording were as described under "Experimental Procedures." The mean change in peak and late currents are discussed under "Results."

View larger version (28K): [in this window] [in a new window]   FIG. 4. Effect of MCh (1 µM) or PMA (100 nM) on the Cav 2.3 currents expressed in Xenopus oocytes with the wild type or the quadruple S888A/S892A/S894A/S1987A mutant of the 1 2.3 subunit. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The drug treatment and the current recording were as described under "Experimental Procedures." The top panels show the current traces, and the bottom panels show the averaged peak and late currents in the wild type and the mutant. Numbers in parenthesis indicate "n." **, p < 0.001; *, p < 0.05, compared with wild type; t test.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Effect of MCh (1 µM) on Cav currents coexpressed with 1, 1B, 2/ subunits and muscarinic M1 receptors in Xenopus oocytes. The oocytes injected with the cDNA were voltage clamped after 7–8 days of incubation at 16 °C. They were held at –80 mV and depolarized to the test potential of 0 mV for all types of Cav channels except Cav 2.2. A test potential of 20 mV was used for Cav 2.2 channels. The drug treatment and the current recording were as described under "Experimental Procedures." The mean change in peak and late currents are discussed under "Results."

We identified nine serine/threonine sites in the ₁ 2.3 subunit (Fig. 3). Four potential phosphorylation sites (Ser-888, Ser-892, and Ser-894 in the II–III linker and Ser-1987 in the C terminus) are unique to the ₁ 2.3 subunit and are considered potential receptor-mediated (M₁) PKC phosphorylation sites, because, among the members of the Ca_v 2.0 family, only Ca_v 2.3 currents were potentiated by MCh application. The other five sites are homologous to the five potential PKC phosphorylation sites we noted in the ₁ 2.2 subunit. This second set of potential phosphorylation sites was considered potentially PMA-selective, because both Ca_v 2.2 and 2.3 currents were potentiated by PMA.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Schematic diagram of Cav channel 1 2.2 and 2.3 subunits showing the predicted potential PKC phosphorylation sites. I–IV indicates the four transmembrane domains. The numbers with the circles show the positions of the amino acids. The sites within the rectangles are homologous. The strategy for selecting these sites is described under "Results."

Although MCh or PMA potentiated the wild type I_Ba, differential results were observed with these agents in the oocytes expressing the quadruple mutation S888A/S892A/S894A/S1987A. MCh-induced potentiation of both the peak and the late I_Ba was significantly decreased in S888A/S892A/S894A/S1987A compared with the wild type. In contrast, the PMA-induced increase in the I_Ba was not affected significantly in the quadruple mutation (Fig. 4, A and B, and Table I).

View larger version (33K): [in this window] [in a new window]   FIG. 5. Effect of MCh (1 µM) on the Cav 2.3 currents expressed with the wild type or the triple mutant, S888A/S892A/S894A of 1 2.3 subunit in Xenopus oocytes. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The drug treatment and the current recording were as described under "Experimental Procedures." The top panel shows the current traces, and the bottom panel shows the averaged peak and late currents in the wild type and the mutant. Numbers in parenthesis indicate "n." **, p < 0.001; *, p < 0.01, compared with wild type; t test.

View larger version (45K): [in this window] [in a new window]   FIG. 6. Summary of the effect of MCh (1 µM) on the Cav 2.3 currents expressed in Xenopus oocytes with the wild type or the single (S888A, S892A, S894A) or double mutation (S888A/S892A, S888A/S894A, S892A/S894A) of II–III linker serine residues of 1 2.3 subunit. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The panels show the averaged peak and late currents in the wild type and the mutants following MCh treatment. Numbers in parenthesis indicate "n."

View larger version (35K): [in this window] [in a new window]   FIG. 7. Effect of MCh (1 µM) on the Cav 2.3 currents expressed in Xenopus oocytes with the wild type, S1987A, or the combined double mutation of II–III linker serine residues and Ser-1987 (S888A/S1987A, S892A/S1987A, S894A/S1987A) of the 1 2.3 subunit. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The panels show the averaged peak and late currents in the wild type and the mutants following the treatment with MCh. Numbers in parenthesis indicate "n." *, p < 0.001, compared with wild type; t test.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Effect of MCh (1 µM) on the Cav 2.3 currents expressed in Xenopus oocytes with the wild type or the triple mutants that included S1987A and any two serine residues of II–III linker (S888A/S892A/S1987A, S888A/S894A/S1987A, S892A/S894A/S1987A) of the 1 2.3 subunit. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The panels show the averaged peak and late currents in the wild type and the mutants following the treatment with MCh. Numbers in parenthesis indicate "n." **, p < 0.01; *, p < 0.05, compared with wild type; t test.

View larger version (42K): [in this window] [in a new window]   FIG. 9. Basal Cav 2.3 currents of the mutants S888D, S892D, S1987D, and S892D/S1987D of the 1 2.3 subunit and the effect of MCh in Xenopus oocytes. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The panels show the averaged peak and late currents in the wild type and the mutants with MCh (1 µM) treatment. Numbers in parenthesis indicate "n." a, p < 0.001; b, p < 0.01; c, p < 0.02; d, p < 0.05, compared with wild type; t test.

View larger version (47K): [in this window] [in a new window]   FIG. 10. Basal Cav 2.3 currents of the mutants S888D, S892D, S1987D, and S892D/S1987D of 1 2.3 subunit and the effect of PMA (100 nM) in Xenopus oocytes. The currents were coexpressed with 12.31B2/ subunits and M1 receptors. The panels show the averaged peak and late currents in the wild type and the mutants with PMA treatment. Numbers in parenthesis indicate "n." **, p < 0.01; *, p < 0.05, compared with wild type; t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Differential Sensitivity of Ca_v Channels to Modulation Related to Various ₁ Subunits—Different Ca_v currents vary in their responses to several hormones, neurotransmitters, and agonists that can activate PKC. The PKC activator PMA failed to modulate Ca_v 1.2c currents, whereas M₁ receptor stimulation, which can activate PKC, decreased the currents through Ca_v 1.2c channels. Neither PMA nor M₁ receptor activation modulated Ca_v 2.1 currents. In contrast, PMA and M₁ receptor activation each potentiated Ca_v 2.3 currents, whereas Ca_v 2.2 currents were increased by PMA only (see Figs. 1 and 2). Because all of these Ca_v channels were expressed in the same oocyte expression system, the same PKC isozymes should be available in all cases. The selective action of MCh on the Ca_v 2.3 currents may be related to unique PKC sites in the Ca_v 2.3 channels. It is likely that the channel-specific effects of MCh or PMA observed here were contributed by the ₁ subunit of these channels, because the auxiliary subunits (1B and 2/) used in the expression of these channels were the same. Hence, the selective action of MCh on Ca_v 2.3 current may be due to the presence of unique phosphorylation sites in the ₁ 2.3 subunit. In agreement with this hypothesis, potential phosphorylation sites of Ser at 888, 892, 894, and 1987 were identified in the ₁ 2.3 subunit, and their quadruple mutation to Ala inhibited the effect of MCh but left the effect of PMA intact under the same conditions (Fig. 4).

Potential PKC Sites in the II/III Linker of the ₁ 2.3 Subunit Are Required for Enhancement of Current by MCh—Serine sites unique to the II/III linker of the ₁ 2.3 subunit seem to be of critical importance for the MCh responsiveness of these channels. Triple Ser Ala mutation of all three of these sites caused loss of the MCh response (see Fig. 5). The observation that double or single mutations in this region did not eliminate the response suggests that any one of the sites, Ser-888, Ser-892, or Ser-894, is sufficient for altered channel gating by M₁ receptor activation (see Fig. 6). However, the sites are not equivalent. If Ser-1987 in the C terminus is mutated to Ala, the II–III linker sites Ser-888 or Ser-892 are still sufficient for the MCh response, but Ser-894 is not (see Fig. 8 and Table I).

The basal current of the channel was significantly increased (at least 2-fold) following the Ser Asp substitution of 888 or 892. In the face of this high basal activity, MCh caused less activation of the current, suggesting that the major effect of the Asp mutations was to enhance channel gating and opening rather than to increase channel expression. In the latter case, MCh stimulation similar to control would have been expected. These results are consistent with the hypothesis that the Ca_v 2.3 channel-specific response of MCh is mediated via these unique sites in the II/III linker.

Several observations suggest that the MCh enhancement may be mediated via phosphorylation of the sites in the II/III linker: 1) The implicated sites were selected because they fit a PKC phosphorylation motif. 2) Replacement of the potentially phosphorylated Ser with Ala eliminates the MCh effect. 3) Single mutation of any of these sites to Asp (to mimic the negative charge of a phosphate) increases basal activity greatly, and there is less stimulation by MCh. 4) The MCh-induced increase in the Ca_v 2.3 currents was inhibited by PKC inhibitors (13).

Ser-1987 in the C Terminus of the ₁ 2.3 Subunit Plays a Minor Role in the Enhancement by MCh—The lack of MCh enhancement, when Ser-1987 is available but when all the II/III linker serines were mutated to alanine (see Fig. 5), indicates that Ser-1987 is not sufficient for the MCh response. The presence of an intact MCh response in the Ser to Ala or Ser to Asp substitution of Ser-1987 (Figs. 7 and 9) indicates that Ser-1987 is not necessary for the MCh response. However, the presence of a serine at position 1987 could enable Ser-894 to serve as the sole available Ser site in the II–III linker (when residues 888 and 892 were mutated to Asp). When residue 1987 also was mutated to Ala, Ser-894 was insufficient alone and Ser-888 was insufficient in the presence of Ser-894. It is possible that Ser-888 and Ser-894 are incompatible when Ser-892 and Ser-1987 are mutated to Ala (see Fig. 7 and Table I). Attempts to make mutant ₁ 2.3 subunits with Ser Asp substitutions at 894 were unsuccessful due to lack of expression of the mutant constructs (data not shown). Collectively, this information suggests that a constitutive negative charge at residue 894 may be detrimental for protein expression. Ser-892 seems to be capable of mediating MCh responsiveness independent of the availability of serine residues at the other sites examined.

The Ca_v 2.3 II–III Linker Sites Are Not Required for Responsiveness to PMA—In contrast to MCh, PMA still increased the Ca_v 2.3 currents when potential PKC sites unique to Ca_v 2.3 channels (Ser-888, Ser-892, and Ser-894 in the II–III linker and Ser-1987 in the C terminus) were mutated to Ala (Fig. 4 and Table I) or to Asp (Fig. 10). The PMA responsiveness may be conferred by different sites such as Ser-369 in the I–II linker, Thr-879 in the II–III linker, and Ser-1995 and Ser-2011 in the C-terminal segment that are homologous to sites Thr-422, Ser-425, Thr-926, Ser-2108, and Ser-2132 in the PMA-responsive Ca_v 2.2 channels. Thr-422 and Ser-425 have been identified as PMA-responsive sites in the ₁ subunit of Ca_v 2.2 channels (5).

Non-selective PKC inhibitors (a PKC pseudosubstrate and high concentrations of Ro-31-8425) blocked both PMA and MCh effects on Ca_v 2.3 channels suggesting involvement of PKCs in responses to both agonists (13). However, inhibitors more selective for calcium-dependent PKC isozymes (a PKC translocation inhibitor or low concentrations of Ro-31-8425) or agents that prevent release or action of intracellular calcium blocked only the MCh response (13). These results are consistent with the hypothesis that MCh activates calcium-dependent PKCs via phospholipase activation to generate diacylglycerol and release calcium from intracellular stores. These calcium-dependent PKCs may phosphorylate specifically the II–III linker sites unique to Ca_v 2.3 channels. PMA can bind to both cPKCs and nPKCs with much greater affinity than do diacylglycerols; but, because it does not generate directly an increase in intracellular calcium, this action may be inadequate to activate cPKCs. PMA alone may activate predominantly nPKCs.

The Ca_v 2.3 channels contribute, along with Ca_v 2.1 and Ca_v 2.2 channels, to action potential-induced Ca influx in the central nervous system and at many peripheral synapses (18–21). The II/III linker in Ca_v 2.1 and Ca_v 2.2 channels is a site of interaction with the soluble N-ethylmaleimide-sensitive attachment factor receptor proteins required for the release of neurotransmitters (22–24). Receptor-induced phosphorylation of the II/III linker in Ca_v 2.3 channels may provide a rapidly reversible mechanism for regulating just the Ca_v 2.3 channel subtype, either directly, by altering intramolecular interactions between channel domains to promote channel opening, or possibly via effects on interaction with other regulatory proteins (G proteins or channel subunits). Thus M₁ receptor-mediated regulation of these II/III linker sites may contribute to regulation of neurotransmission in both the central and peripheral nervous systems.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants GM65214 (to G. L. K.) and GM31184 (to J. J. S.) and by the Department of Anesthesiology, University of Virginia Health Sciences Systems. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 Anesthesiology, P. O. Box 800710, University of Virginia Health Sciences System, 1766 Lane Rd., Charlottesville, VA 22908-0710. Tel.: 434-924-2924; Fax: 434-982-0019; E-mail: gk3p{at}virginia.edu.

¹ The abbreviations used are: Ca_v, voltage-gated Ca²⁺ channel; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; cPKC, classic PKC; nPKC, novel PKC; aPKC, atypical PKC; DAG, diacylglycerol; MCh, acetyl--methylcholine.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. T. P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada) for the clones of Ca channels and Dr. T. I. Bonner (Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD) for the muscarinic M₁ receptor clone. The technical assistance of Jacqueline Washington is gratefully appreciated.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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