Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C

Brain Research 968 (2003) 227–237 www.elsevier.com / locate / brainres

Research report

Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C Ganesan L. Kamatchi*, Shveta N. Tiwari, Carrie K. Chan, Daguang Chen, Sang-Hwan Do 1 , Marcel E. Durieux 2 , Carl Lynch III Department of Anesthesiology, P.O. Box 800710, University of Virginia Health Sciences Systems, Charlottesville, VA 22908 -0710, USA Accepted 6 January 2003

Abstract Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca v ; with a 1 b1Ba2 / d subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M 1 muscarinic receptor activation in Xenopus oocytes. The inward Ca 21 -current with Ba 21 (IBa ) as the charge carrier was potentiated by PMA or acetyl-b-methylcholine (MCh). The inactivating [I( inact ) ] and non-inactivating [I(noninact ) ] components of IBa and the time constant of inactivation t( inact ) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or b-pseudosubstrate. MCh effect was blocked by atropine, guanosine-59-O-(2-thiodiphosphate) trilithium (GDPbS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca 21 ([Ca 21 ] i ) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and bC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, bC2.4 or Ro-31-8425 is suggestive of the involvement of Ca 21 -dependent cPKC in MCh action, whereas cPKC and Ca 21 -independent novel PKC (nPKC) in PMA action. PMA led to additional increase in IBa that was already potentiated by preadministered MCh (1 or 10 mM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the a 1 2.3 subunit of Ca v 2.3 channels.  2003 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Calcium channel physiology, pharmacology and modulation Keywords: Calcium channel; Muscarinic receptor; Protein kinase C; Xenopus oocyte

1. Introduction Voltage-gated Ca (Ca v ) channels are a large family of heteromultimeric proteins containing a pore forming a 1

*Corresponding author. Tel.: 11-434-924-2924; fax: 11-434-9820019. E-mail address: [email protected] (G.L. Kamatchi). 1 Present address: Department of Anesthesiology, Seoul National University Medical College, Yeongon-dong, Chongno-ku, Seoul, South Korea 110-744. 2 Present address: Department of Anesthesiology, University Hospital Maastricht, P. Debyelaan 25, Maastricht, The Netherlands.

subunit and auxiliary b, a2 / d and g subunits. The a 1 subunit consists of four domains, each with six transmembrane (TM) segments and determines the respective class of Ca v channels since it has the agonist and antagonist binding sites. Electrophysiological and pharmacological characterizations have determined that a 1 1.1, 1.2, 1.3, 1.4 subunits encode L-type channels, a 1 2.1 encodes P/ Qtype channels, a 1 2.2 encodes N-type channels, a 1 2.3 encodes R-type channels and a 1 3.1, 3.2, 3.3 encode T-type of Ca v channels, respectively [10,17]. Some of these Ca v channels are modulated differentially by intermediaries such as G protein bg subunits, intracellular Ca 21 ([Ca 21 ] i ) or protein kinase C (PKC) [13,18,22,31]. PKC appears to play a dominant role as the upregulation induced by PKC antagonized G-protein-mediated inhibi-

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02245-5

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tion of expressed Ca v 2.2 currents [13]. Furthermore, PKC participates in a variety of cellular functions including signal transduction, receptor modulation, vasoconstriction and gene expression [6,11,37,39]. Eleven PKC isozymes (a, bI, bII, g, d, ´, j, h, u, l and m) have been identified so far and they differ in structure and requirements for activation by diacylglycerol (DAG) and Ca 21 [23,27]. These isozymes appear to target a 1 subunits of Ca v channels even though the auxiliary b and a2 / d subunits also possess multiple consensus sites for phosphorylation, and several of these subunits have been shown to be phosphorylated in vitro by PKC [15,21,26,31]. However, all the expressed Ca v currents did not respond similarly to PKC activation in spite of the predicted presence of PKC phosphorylation sites in all types of Ca v channel a 1 subunits. For example, Ca v 2.2 or 2.3 currents, expressed with b1b subunit, were potentiated by PKC activation induced by phorbol 12-myristate 13-acetate (PMA) whereas Ca v 1.2c or 2.1 currents were unaffected under the same conditions [31]. The presence of PKC phosphorylation sites in Ca v channel subunits suggests that Ca v channels may represent a potential target for some hormones, neurotransmitters and agonists that activate PKC. Prominent among these are the odd numbered (M 1 , M 3 , and M 5 ) muscarinic receptors, whose stimulation culminates in the activation of PKC [5]. Muscarinic receptors are prevalent in hippocampus, dentate gyrus, amygdala and cortex [2]. These brain regions also express a 1 2.3 subunits [24,30,32,34,38]. In addition Ca v 2.3 channels share similar anatomical localization with M 1 receptors in the neocortex, neostriatum, CA1–CA3 pyramidal cells, dentate granule cells, etc. [20,32,38]. This correlation in distribution suggests that M 1 receptors may influence cytosolic calcium levels and calcium-dependent electrical events in the neurons of the above CNS regions through Ca v 2.3 channels. Such an action is supported by the observation that currents through Ca v 2.3 channels was increased by M 1 receptor activation in a PKC-dependent manner [19,22]. It is possible that differential PKC isozymes are involved in the potentiation of Ca v 2.3 currents induced by M 1 receptor activation or by PMA. We observed that the potentiation of Ca v 2.3 currents induced by PMA was totally blocked by volatile anesthetics whereas acetyl-b-methylcholine (MCh)-induced increase in Ca v 2.3 currents was only partially inhibited [19]. Based on the differential sensitivity of PKC-induced modulation of Ca v 2.3 currents to anesthetics we hypothesize that various PKC isozymes may be involved in the regulation of Ca v 2.3 channels. We tested this hypothesis by coexpressing M 1 receptors and Ca v 2.3 channels in Xenopus oocytes, a convenient assay system for investigating the modulation of cloned ion channels and receptors. Endogenously they express the required constituents for receptor activation including G-proteins, phospholipase C (PLC) and PKC [7,29].

2. Materials and methods

2.1. Harvesting of oocytes and cDNA injection Mature female Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI, USA), housed in an established frog colony, and fed regular frog brittle twice weekly. For the removal of oocytes, a frog was anesthetized in 500 ml of 0.2% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO, USA) 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 mM: NaCl 82.5, KCl 2, MgCl 2 1.8, HEPES 5, pH 7.5) and transferred to OR2 solution containing 1 mg / ml of collagenase (type 1A; Sigma). The dish containing the oocytes in collagenase solution was agitated for a period of 2–3 h at room temperature in order 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 mM: NaCl 88, KCl 1, NaHCO 3 2.4, CaCl 2 0.41, MgSO 4 0.82, HEPES 15, pH 7.4) containing 2.5 mM sodium pyruvate and 10 mg / ml gentamycin sulfate. The oocytes were allowed to recover by incubation at 16 8C for 3–10 h before cDNA injection. Nuclear (germinal vesicle) injection was performed (Drummond ‘Nanoject’, Drummond Scientific, Broomall, PA, USA) using a maximum of 4 ng of cDNA containing 3 ng of a 1:1:1 mix (molar ratio) of rat brain Ca v a 1 2.3b1Ba2 / d cDNA subunits in pMT2 vector [31] and 1 ng of rat M 1 receptor cDNA in pcDNA 3.1 (Invitrogen, Carlsbad, CA, USA). For the expression of single or two subunits of Ca v 2.3 channels, 3 ng of a 1 2.3 subunit alone or equimolar amounts of a 1 2.3b1B or a 1 2.3a2 / d subunits to a concentration of 3 ng along with 1 ng of M 1 receptor were employed. The oocytes were returned to Barth’s solution and incubated at 16 8C for 6–8 days before the recording of current.

2.2. Current recording Macroscopic currents, with Ba 21 (IBa ) as the charge carrier, were recorded employing a two-electrode voltageclamp technique using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA, USA). The amplifier was linked to interface and an IBM-PC-compatible computer equipped with pClamp software (version 5.6; Axon Instruments) for data acquisition. Leak currents were subtracted using the P/ 4 procedure. Microelectrodes were filled with

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3 M CsCl; typical resistances were 0.5 to 2.5 MV. KClAgar 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 ml volume) superfused with the recording solution containing (in mM): 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 280 mV before being depolarized to 0 mV test potential for a period of 850 ms. To construct the current– voltage (I–V ) relationship for Ca v 2.3 currents, 450 ms test potentials were employed starting from 250 to 100 mV with incremental steps of 10 mV.

2.3. Drug treatment All of the oocytes exhibiting IBa greater than 400 nA underwent control, treatment and wash protocol. The control IBa was recorded at the 8th min after the oocyte was impaled. MCh was used to activate M 1 receptors. The effect of PMA was also tested in oocytes coexpressing M 1 receptors. MCh or PMA was perfused for 30 s and the current was recorded after 90 s from this period, thus exposing the oocyte to the agonist for a period of 2 min. The effect of MCh was recorded at 2 and 4 min (as control) in the experiments involving the sequential administration of MCh or PMA. The involvement of M 1 receptors and the various second messengers such as Gproteins, PLC, inositol-1,4,5-triphosphate (IP3), [Ca 21 ] i and PKC in M 1 muscarinic receptor-induced modulation of Ca v 2.3 currents was studied with the use of blockers of the respective intermediaries. In case of injection of any of these agents into the oocytes, the intracellular concentration was calculated based on the oocyte volume (|1 ml) and the solution injected. A batch of oocytes was pretreated with atropine (1 mM) for a period of 5 min before challenging with MCh in order to demonstrate the involvement of M 1 receptors. The role of G-proteins was tested with the intracellular injection of guanosine-59-O-(2thiodiphosphate) trilithium (GDPbS; 10 mM; 50.6 nl) at least 30 min before the recording of current. Involvement of PLC was examined by incubating the oocytes in 2 mM U-73122 at room temperature for 40 min to 1 h before testing with MCh. Low molecular weight (|3000 Da) heparin (2 mM; 50.6 nl) was injected intracellularly to block IP3 receptors 30 to 60 min before current recording. To study the role of [Ca 21 ] i , BAPTA tetrasodium solution (40 mM; 41.4 nl) was microinjected into the oocyte 1–3 h before clamping. The involvement of PKC was examined by 20 min perfusion of oocytes with Ro-31-8425 (25 nM for MCh and 500 nM for PMA), a bisindolylmaleimide that has lower IC 50 for conventional PKCs (cPKC) and higher IC 50 for novel PKCs (nPKC) before challenging with MCh or PMA. The specific contribution of cPKCs

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and nPKCs in the action of MCh and PMA was examined with the use of the peptide bC2.4 that inhibits translocation of cPKC or b-pseudosubstrate that inhibits the activity of cPKCs and nPKCs. These agents were injected intracellularly (5 mM; 41.4 nl leading to a final concentration of |200 nM) about 5 h before challenging with MCh or PMA. When testing with the peptides bC2.4 or b pseudosubstrate, a low concentration (10 nM) of PMA was employed as suggested by their supplier (Dr. MochlyRosen laboratory, Stanford University, Stanford, CA, USA). A sequential application technique was employed in order to examine the contribution of different types of PKCs in the action of MCh and PMA. In these experiments MCh (1 or 10 mM) was administered first and its response was recorded at 2 min. This was followed immediately with the perfusion of a combination of MCh and PMA for a period of 30 s (i.e. till 2:30 min). The currents were then recorded at 4 min (i.e. 2 min from the beginning of perfusion of drug combination).

2.4. Chemicals PMA, 4a-phorbol 12,13-didecanoate (4a-PDDC), Ro31-8425 (Calbiochem, San Diego, CA, USA) and U-73122 (RBI, Natick, MA, USA) were dissolved in DMSO (0.1%). GDPbS (RBI), MCh, niflumic acid, heparin (Sigma) and BAPTA tetrasodium (Calbiochem) were dissolved in distilled water. bC2.4 and b-pseudosubstrate (Dr. Mochly-Rosen laboratory) were diluted in water. All of these agents except niflumic acid were prepared as concentrated stock solutions and stored frozen at 220 8C. They were diluted to their final concentration in recording solution on the day of the experiment. To block endogenous Cl 2 currents, niflumic acid was added to the recording solution, which was stirred overnight in order for it to dissolve.

2.5. Data analysis The data are shown as means6S.E.M., 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 employed as a measure of relative degree of channel inactivation. The data were analyzed using either the PCS program [25] or Clampfit, version 6.0.2 (Axon Instruments). Ba 21 conductance (GBa ) was calculated as IBa /(V 2Vrev ) using the reversal potential (Vrev ), as determined by interpolation over the voltage step at which current changed from inward to outward. The voltage dependence of activation of GBa was then described by a Boltzmann equation of the form: GBa 5 GBa,max h1 1 exp [(V 2Vn ) /k n ]j 21 ,

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where Vn is the voltage of half-maximal activation and k n is the slope factor determined from a least squares fit (Sigma Plot, Jandel Scientific Corp., San Rafael, CA, USA). The inactivating component of IBa was described by a single exponential component with R 2 values consistently exceeding 0.98 for a least squares fit. This behavior was described by the formula: IBa 5 I(inact ) exp (2t /tinact ) 1 I(noninact ) , where I(inact) is the inactivating current, t(inact ) is the time constant of inactivation and I(noninact ) is the residual noninactivating IBa . Since descriptions employing two exponential components rarely provided improvements in the fit as determined by the coefficient of variation (R), only a single exponential was employed as reported previously when a 1 subunits were expressed with the b1B subunit [40]. Statistical significance was determined using Dunnett’s, paired or unpaired t-test and P,0.05 was considered significant.

3. Results About 80% of the oocytes injected with the cDNA expressed the Ca v 2.3 currents. These currents appeared at 230 mV and reversed between 50 and 70 mV when the oocytes expressing Ca v 2.3 channels underwent depolarizations (by 10 mV increments) from 250 to 100 mV. The peak amplitude was observed between 210 and 10 mV, at 6564.8 ms (mean6S.D.; n556). This behavior is similar to that described when cloned neuronal Ca v 2.3 channels from rat [30], rabbit [32], mouse or human brain [34] were expressed in a similar system.

3.1. Effect of MCh or PMA on the behavior of Cav 2.3 currents Expressed Ca v 2.3 currents were increased by either MCh (1 mM) or PMA (500 nM) whereas 4a-PDDC failed to significantly influence the IBa (Fig. 1). Although PMA or MCh affected both the peak and late IBa their influence on the latter was proportionally greater than that on the peak current. The concentration–response relationship for effects of MCh on the peak Ca v 2.3 current is shown in Fig. 2A. Data were fitted to the Hill equation, and the Hill coefficient was calculated to be 1.11, suggesting a single binding site. The EC 50 for MCh was calculated to be 1.16 mM; 1 mM MCh was used for the bulk of the study to provide a moderate response. I–V plots for peak IBa through Ca v 2.3 channels for control, MCh treatment and recovery were constructed and the mean values for six experiments each are shown in Fig. 2B. The reversal potential was unchanged (64.861.69, 64.561.43 and 63.561.99 mV for the control, MCh and recovery, respec-

Fig. 1. Effects of MCh, PMA or 4a-PDDC on Ca v 2.3 currents coexpressed with a 1 2.3b1Ba2 / d subunits and muscarinic M 1 receptors in Xenopus oocytes. The oocytes injected with the cDNA were voltage clamped after 7–8 days of incubation. They were held at 280 mV and depolarized to 0 mV. The control IBa was recorded at 8 min after the oocyte was impaled. This was followed by the perfusion of MCh (A) or PMA (B) or 4a-PDDC (C) for 30 s and the current was recorded after 90 s, thus testing the oocyte 2 min after the beginning of perfusion with these agents. (D) Summary for the mean values for MCh, PMA or 4a-PDDC. a P,0.001, b P,0.02, compared to the respective control; paired t-test.

tively). While the mean peak IBa for control and recovery was observed between 210 and 0 mV, MCh shifted the peak to more hyperpolarizing potentials. Analysis of the conductance revealed that MCh shifted the conductance to hyperpolarizing potentials (Fig. 2C). The Vn for control, MCh and recovery were 222.161.7, 227.661.1 and

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Fig. 2. Characterization of MCh-induced potentiation of Ca v 2.3 currents in Xenopus oocytes coexpressed with a 1 2.3b1Ba2 / d subunits and M 1 receptors. (A) The concentration–response relationship for the peak current was determined with various concentrations of MCh. The oocytes were activated to 0 mV from a holding potential of 280 mV. The response in the presence of MCh was recorded 2 min after its perfusion. Every oocyte was used for testing one concentration only; n53–9. (B) I–V plots for control, MCh and wash were constructed with incremental steps of 10 mV from 250 to 100 mV. The averaged reversal potential for control, MCh and recovery may be seen under the results (n56). (C) The averaged conductance was determined for the same current tracings shown in (B). The normalized conductance was determined and fitted to a Boltzmann equation as mentioned in Section 2.5. Inset: averaged Vn and k n for the control, MCh and wash. The values of S.E.M. for Vn and k n are provided in Section 3. (D) Effects of MCh or PMA on the various components of IBa through a 1 2.3b1Ba2 / d-channels. *P,0.001, compared to control, using paired t-test; a P,0.01, compared to I( inact ) using t-test.

220.862.3 mV, respectively, a significant hyperpolarizing shift of 5.5 mV with MCh (P50.02 vs. control). The k n (slope factor) for control, MCh and recovery were 5.060.34, 3.860.68 and 5.460.16 mV, respectively (Fig. 2C), a difference that was not statistically significant. The kinetic behavior of inactivation of IBa through Ca v 2.3 currents was examined based on the increase in late current with PMA or MCh. I(noninact ) and t(inact ) (for PMA: 198616 vs. 335617 ms; n58; for MCh: 189614 vs. 218614 ms; n515) were both increased consistent with the slowing of inactivation. The increase in the I(noninact ) component of Ca v 2.3 currents was significantly greater than that of I(inact) with either PMA or MCh. However, PMA-induced increase in t(inact) was much pronounced compared to MCh (Fig. 2D). The basic properties of IBa differed widely when a 1 2.3 subunit was expressed alone, or in combination with either b1B or a2 / d subunit (Fig. 3). The amplitude of IBa through these channels was in the order of a 1 2.3, a 1 2.3a2 / d,a 1 2.3b1B as suggested before [31]. In oo-

cytes expressing the a 1 2.3 subunit alone or a 1 2.3a2 / d, MCh increased the late IBa without significantly affecting peak IBa . However, when the b1B subunit was expressed with the a 1 2.3 subunit, the peak current was increased in addition to late IBa (Fig. 3).

3.2. Pharmacology of MCh or PMA-induced potentiation of Ca v 2.3 current MCh failed to potentiate IBa after treatment with atropine, suggesting the involvement of muscarinic receptors and ruling out the possibility of any non-specific activity. Similarly, after the treatment with GDPbS or U-73122, the inhibitors of G-proteins and PLC, respectively, MCh failed to increase significantly the IBa through Ca v 2.3 channels (Fig. 4). Furthermore, MCh-induced potentiation of Ca v 2.3 current was eliminated in the oocytes injected with either heparin (a blocker of IP3 receptors), BAPTA (to chelate Ca 21 ) or bC2.4 (inhibitor of translocation of cPKC) (Fig. 5). However, PMA still potentiated both

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components of IBa in oocytes treated with heparin, BAPTA or bC2.4 (Fig. 5). Either MCh or PMA failed to potentiate the Ca v 2.3 current in oocytes injected with b-pseudosubstrate, a peptide known to inhibit the activity of both cPKC and nPKC (Fig. 6). Similarly, the effect of MCh or PMA was blocked after the treatment of the oocytes with Ro-31-8425, a selective inhibitor of PKC. A concentration of 25 nM was sufficient to block MCh whereas 500 nM was required to block the effect of PMA (Fig. 6).

3.3. Different PKC isozymes may be involved in the potentiation of Ca v 2.3 currents

Fig. 3. Effect of MCh on the a 1 2.3 subunit Ca v channel expressed alone or in combination with auxiliary b1B or a2 / d subunits. All these oocytes were injected with 1 ng of M 1 receptor cDNA in addition to 3 ng of Ca v channel subunits. These oocytes were held at 280 mV and depolarized to 0 mV. Following the control measurements, MCh was perfused for a period of 30 s and the IBa was recorded at 2 min. (A) IBa through Ca v 2.3 channels expressed with a 1 2.3 subunit only. Three ng of a 1 2.3 subunit cDNA were injected as mentioned in Section 2. (B) IBa through Ca v 2.3 channels expressed with a 1 2.3b1B subunits. Equimolar concentration of a 1 2.3 and b1B subunits not exceeding 3 ng was injected in these oocytes. (C) IBa through Ca v 2.3 channels expressed with a 1 2.3a2 / d subunits. Equimolar concentration of a 1 2.3 and a2 / d subunits not exceeding 3 ng was injected in these oocytes. (D) Summary for the mean effect of MCh on the peak and late IBa shown at A, B and C above. Numbers in parentheses indicate ‘n’. a P,0.001, b P,0.01, c P,0.02, d P,0.05, compared to the respective control; paired t-test.

Administered alone, MCh (1 mM) increased both the peak and late current at 2 (4665.1 and 5967.2%) and 4 min (2565.3 and 2468.6%; n55), respectively (Fig. 7 Ai and Aii). There was additional increase (compared to MCh 4 min) in the peak and late current when PMA and MCh (1 mM) were administered together after MCh (1 mM). The increase in the peak and late current produced by the combined administration of PMA and MCh (1 mM) at 4 min was significantly different from that of the MCh 4 min value (Fig. 7 Aii vs. Bii). Similar results were obtained when a higher concentration of MCh (10 mM) was followed by the combination of PMA and MCh (10 mM). Administered alone, MCh (10 mM) increased both the peak and late current at 2 (9769 and 175637%) and 4 min (4767 and 86632%; n55), respectively (Fig. 7 Ci and Cii). When the higher concentration of MCh was followed by the combination of PMA and MCh, the peak and late current at 4 min further increased to 88612 and 172618%, respectively. The increase in the peak and late current produced by MCh and PMA was significant when compared to MCh alone at 4 min (Fig. 7 Cii vs. Dii). Collectively these experiments suggest that the effects of M 1 receptor activation on Ca v 2.3 currents are mediated through the G-proteins, PLC, IP3 receptors, [Ca 21 ] i and PKC. These findings are consistent with a prominent role for PKC since PMA duplicated the effects of M 1 receptor activation. The differences in the effects of MCh and PMA after the blockade of IP3 receptors, chelating [Ca 21 ] i or the inhibition of translocation of cPKCs may suggest the involvement of specific PKC isozymes in the responses.

4. Discussion The physiological consequences of activation or inhibition of Ca v currents may be considerable, particularly in the nervous system. The modulation of Ca v currents may be initiated when the intracellular second messengers such as G protein bg subunits, [Ca 21 ] i , PKC, etc., bind with their recognition sites in the subunit components of Ca v channels [13,18,22,31]. In this study we examined the potentiation of Ca v 2.3 currents by the activation of PKC directly with PMA and indirectly through activating the

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Fig. 4. Effects of atropine, GDPbS (G-protein inhibitor) or U-73122 (PLC inhibitor) on Ca v 2.3 currents coexpressed with a 1 2.3b1Ba2 / d subunits and M 1 receptors in Xenopus oocytes. The response in the presence of MCh was recorded 2 min after its perfusion. (A) The oocytes were pretreated with atropine (1 mM) for a period of 5 min before challenging with MCh. (B) GDPbS (10 mM; 50.6 nl) was injected intracellularly at least 30 min before testing the response of MCh. (C) Oocytes were incubated with U-73122 (2 mM) at room temperature for 40 min to 1 h to inhibit PLC before testing with MCh. (D) Summary of the experiments, A to C. Numbers in parentheses indicate ‘n’.

G-protein pathway; the muscarinic M 1 receptor system was used as a model or surrogate for G-protein-mediated pathway. It is well-known that the activation of G-proteins modulated various Ca v channels [9,17]. It has been reported that the bg subunits of G proteins bound to a 1 2.1 or 2.2 subunits leading to the inhibition of the current [14,16]. The modulation of Ca v 2.3 currents by G-proteins was variable, depending in part upon whether a b subunit is coexpressed with the a 1 2.3 subunit [9,28]. Interaction of the bg subunits of G-proteins with the I–II loop of a 1 2.1, 2.2 and 2.3 subunits (but not with a 1 1.2), within the binding site described for the Ca v channel b subunit, appears to mediate G-protein-induced modulation of these channels [9]. Taken together the above studies suggest a direct action of G-proteins on the Ca v channels. However, in the present study, G-protein appears to be an intermediary in a cascade of events that culminate in the activation of PKC and the subsequent potentiation of Ca v 2.3 currents (Fig. 1). The blockade not only of G-proteins, but also of signaling intermediaries of the PKC pathway downstream prevented potentiation of Ca v 2.3 currents by MCh (Figs. 4–6). These effects argue that a G-proteinmediated second messenger pathway is involved in the modulation of Ca v 2.3 currents by M 1 receptor activation.

It has been suggested that the G aq subunit mediated the PTX-insensitive stimulation of PLC seen with M 1 receptor activation [22]. In our study the involvement of PLC in MCh-induced potentiation of Ca v 2.3 currents is supported by the blockade of M 1 receptor activation by U-73122, an inhibitor of PLC (Fig. 4). The subsequent links in this particular G-protein-mediated M 1 receptor pathway appear to be DAG and IP3 (the PLC-induced hydrolysis products of membrane phosphatidylinositol-4,5-bisphosphate), since blockade of any one of these intermediaries inhibited the effect of MCh. Administration of MCh shifted the voltage-dependent opening of Ca v 2.3 channels to a more negative potential. In addition, MCh caused a significant increase in I(inact ) , t( inact ) , and I(noninact ) (Fig. 2). Since PMA also increased I( inact ) , t( inact ) , and I(noninact ) through Ca v 2.3 channels (Fig. 2), it is suggested that the stimulation of M 1 receptors follow a downstream pathway that culminate in the activation of PKC. The suppression of MCh- or PMAinduced increase of Ca v 2.3 currents by the PKC antagonist Ro-31-8425 or b-pseudosubstrate, inhibitor of the activity of cPKC and nPKC supports the above observation that PKC may be the common mediator in the action of these agents (Fig. 6). A lower concentration (25 nM) of

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Fig. 5. Effects of MCh or PMA on Ca v 2.3 currents coexpressed with a 1 2.3b1Ba2 / d subunits and M 1 receptors after the treatment with heparin, BAPTA or bC2.4 (cPKC translocation inhibitor) in Xenopus oocytes. The response in the presence of MCh or PMA was recorded 2 min after its perfusion. (A) Low molecular weight (|3000) heparin (2 mg / ml; 50.6 nl) was injected intracellularly to block IP3 receptors 30 to 60 min before testing MCh or PMA. (B) BAPTA tetrasodium solution (40 mM; 41.4 nl) was microinjected into the oocytes 1–3 h before clamping which was followed by MCh or PMA. (C) bC2.4 was microinjected (5 mM; 41.4 nl) into the oocytes |5 h before clamping which was followed by MCh or PMA (10 nM); note that a low concentration of PMA (10 nM) was employed following the peptide bC2.4 as suggested by its supplier. (Application of PMA (10 nM) showed an increase of 3262 (P,0.001; n55) and 125625% (P,0.01; n55) of peak and late Ca v 2.3 currents, respectively, in control experiments; not shown.) (D) Summary of mean changes in experiments A to C. Numbers in parentheses indicate ‘n’. a P,0.01, b P,0.02, c P,0.05 compared to control; paired t-test.

Ro-31-8425 was sufficient to inhibit MCh response whereas much higher concentration (500 nM) was required to depress the effect of PMA. Ro-31-8425 distinguishes between different PKC isozymes as the IC 50 required to inhibit cPKC isozymes is small whereas a higher IC 50 is required for nPKC isozymes [33]. The requirement of different concentrations of Ro-31-8425 to block the responses of MCh and PMA is an indication for the involvement of various PKC isozymes in the modulation of Ca v 2.3 currents. While PMA can activate a number of PKC isozymes, Ca 21 is a required cofactor only for the cPKC isozymes (a, bI, bII, g) [23,27]. Since, the buffering of [Ca 21 ] i by BAPTA or the blockade of IP3 receptors (whose activation is known to release stored Ca 21 ) with heparin blocked the effect of MCh on Ca v 2.3 channels, it is possible that a member(s) of cPKC is behind the potentiation of Ca v 2.3 currents by M 1 receptor activation (Fig. 5). This is supported by the observation that bC2.4, a peptide inhibitor of cPKC translocation blocked the effect of MCh but not PMA (Fig. 5). PMA further increased the current when it was administered after either 1 or 10 mM MCh (Fig. 7). The additional

increase in the current by PMA after the lower concentration of MCh may either be due to cPKC or nPKC isozymes (d, ´, h, u), since PMA is capable of activating both classes of PKC. It is possible that there are nPKC phosphorylation sites in the a 1 2.3 subunit since PMA still increased the current after the inhibition of cPKC with BAPTA or heparin or bC2.4 (Fig. 5). This is supported in the experiment that involved sequential administration of higher concentration of MCh (10 mM) and PMA. In this experiment PMA further increased the current when it was administered after the potentiation induced by higher concentration of MCh (Fig. 7). Assuming that the high concentration of MCh resulted in phosphorylation of all cPKC susceptible sites, the further increase in current by PMA suggests that there are phosphorylation sites that are specific for nPKC. Studies involving site-directed mutation of selected serine / threonine of the a 1 2.3 subunit have shown that mutations of predicted phosphorylation sites result in differential loss of current enhancement by MCh or PMA (unpublished observation). Though the mechanism of (PKC-dependent) phosphorylation-induced enhancement of Ca 21 -current is not clear, an increase in either

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Fig. 6. Effects of MCh or PMA on Ca v 2.3 currents expressed with a 1 2.3b1Ba2 / d subunits and M 1 receptors after the treatment with Ro-31-8425 or b-pseudosubstrate in Xenopus oocytes. The response in the presence of MCh or PMA was recorded 2 min after its perfusion. (A) Following the control measurements, the oocyte was exposed to Ro-31-8425 (25 nM) for a period of 20 min in the recording chamber, followed by the perfusion of MCh or PMA. (B) Following the control measurements, the oocyte was exposed to Ro-31-8425 (500 nM) for a period of 20 min in the recording chamber, followed by the perfusion of PMA. (C) b-Pseudosubstrate solution was microinjected (5 mM; 41.4 nl) into the oocytes |5 h before clamping which was followed by MCh or PMA (10 nM); note that a low concentration of PMA (10 nM) was employed after b-pseudosubstrate as suggested by its supplier. (See legend for Fig. 5 for details about control response with PMA (10 nM).) (D) Summary of mean changes in experiments A to C. Numbers in parentheses indicate ‘n’. **P,0.01, *P,0.01, compared to control; paired t-test.

channel number, conductance or channel open time has been suggested [12]. Others have postulated, based on single channel studies, that calcium channels may exist in different states that are more or less excitable [1,8], and that the action of PKC is to switch the channel to a more easily excited state [36]. It seems likely that the a 1 subunits of Ca v channels accounted for the differing sensitivity to PKC activation; however, the presence of auxiliary subunits can dramatically alter the expression and behavior of the Ca v currents [31]. In the present study, inclusion of the a2 / d subunit with the a 1 2.3 subunit did not contribute much to the amplitude of current or the response to MCh. In contrast, inclusion of the b1B subunit significantly increased the amplitude of 2.3 current and MCh stimulation of both the peak and the late components of IBa (Fig. 3). As seen here with MCh, PMA also failed to potentiate peak IBa when a 1 2.3 subunit was expressed alone [31]; these authors also found that the inclusion of b1B subunit with a 1 (2.2 or 2.3) potentiated the effect of PMA. It was suggested that the presence of a b subunit is necessary for the phosphorylation of the a 1 subunit, or that the b subunit itself undergoes PKC-induced phosphorylation [3,4]. Yet in the presence or absence of the a2 / d subunit, stimulation of M 1

receptors caused an increase in the late IBa . This result would suggest that at least one site of PKC action on a 1 2.3 requires the presence of the a2 / d subunit. One possibility is that the MCh effect on peak IBa may be due to some altered interaction between a 1 2.3 and b1B subunits, while a direct a 1 2.3 subunit phosphorylation may have resulted in an alteration of the inactivation kinetics. Since cholinergic receptors are involved in higher brain functions such as learning and memory as well as in pathological states like Alzheimer’s disease [20], M 1 receptor-induced modulation of Ca v 2.3 currents is likely to be physiologically important. Ca v 2.3 channels deserve special recognition, since they are located predominantly in the CNS, resistant to classic Ca v channel blockers and involved in the release of neurotransmitters [35,38]. Ca v 2.3 channels share similar anatomical localization with M 1 receptors in CNS regions such as hippocampus, neocortex, neostriatum, CA1–CA3 pyramidal cells, dentate granule cells, etc. [20,32,38]. Hence the activation of M 1 receptors may lead to a significant influence on calcium-dependent electrical events in the cell bodies of the above CNS regions through Ca v 2.3 channels. The evidence for involvement of cPKCs as a link between M 1 receptors and Ca v 2.3 channels suggests that PKC isozymes may be a

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Fig. 7. Time course with MCh (1 or 10 mM) and sequential administration of MCh and PMA on Ca v 2.3 currents coexpressed with a 1 2.3b1Ba2 / d subunits and M 1 receptors in Xenopus oocytes. (Ai) Following 30 s perfusion, the effect of MCh (1 mM) was recorded at 2 and 4 min. (Aii) Summary of MCh-induced potentiation at 2 and 4 min. *P,0.05, compared to control; NS, not significant. Dunnett’s t-test (n55). (Bi) MCh (1 mM) was administered first for a period of 30 s and its effect was recorded at 2 min; this was followed immediately with the perfusion of a solution containing PMA1MCh (1 mM) and the current was recorded at the 4th min. (Bii) The effect of MCh alone and its combined administration with PMA are summarized. a P,0.001, d P,0.05, compared to control; paired t-test (n55). [ P,0.01,@ P,0.02, compared to MCh 4 min shown in Aii; t-test (n55). (Ci) Following 30 s perfusion, the effect of MCh (10 mM) was recorded at 2 and 4 min. (Cii) Summary of MCh-induced potentiation at 2 and 4 min. *P,0.05, compared to control; NS, not significant. Dunnett’s t-test (n55). (Di) MCh (10 mM) was administered first for a period of 30 s and its effect was recorded at 2 min; this was followed immediately with the perfusion of a solution containing PMA1MCh (10 mM) and the current was recorded at the 4th min. (Dii) The effect of MCh alone and its combined administration with PMA is summarized. a P,0.001, compared to control; paired t-test (n55). [ P,0.05 compared to MCh 4 min shown in Aii; t-test (n56).

useful target for pharmacological regulation of selective Ca v channels.

Acknowledgements We are grateful to Dr. T.P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada) for sup-

plying clones of Ca channels and Dr. T.I. Bonner (Laboratory of Cell Biology, National Institute of Mental Health, NIH, Bethesda, MD, USA) for the muscarinic M 1 receptor clone. We thank Drs. J.J. Sando and D. Bayliss for reading this manuscript. The technical assistance of Mrs. Jacqueline Washington is gratefully appreciated. This work was supported by National Institutes of Health Grants to MED (R29-GM52387) and to CLIII (GM31144).

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