Protein Kinase A Activity Controls the Regulation of T-type CaV3.2 Channels by Gβγ Dimers*

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gβγ dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gβ2γx dimers to effect voltage-independent inhibition of Cav3.2 channels. Inhibition requires phosphorylation of Ser1107, a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gβ2γ2 dimers but does not disrupt dimer binding nor change its specificity. Gβγ dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Cav3.2 whole cell current is precluded by Gβγ-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D1 or D2 agonists do not elicit channel inhibition yet together synergize to inhibit Cav3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Cav3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

G-protein ␤␥ dimers released upon receptor activation play a central role in regulating the activity of high voltage-gated calcium channels (HVA) 3 of the Ca v 2 family (N-type, P/Q-type, R-type) by directly interacting with intracellular domains of these channel pore proteins (1)(2)(3)(4)(5)(6). Inhibition of Ca v 2 channels by G␤␥ dimers depends on voltage and can be modulated by protein kinase C (7), and synaptic protein interactions (8) allowing for a range of Ca v 2 channel activity in neurons and neuroendocrine cells, and for the opportunity for presynaptic modulation.
By contrast, LVA channels of Ca v 3 family have a less restricted distribution (9) and are expressed in peripheral tissues (10). They are prominently expressed in zona glomerulosa cells (ZG) of the adrenal gland (11) where Ca v 3.2 channel activity controls the production of aldosterone. LVA channel currents are inhibited consistently by a host of GPCRlinked hormones (dopamine, enkephalin, nociceptin, bradykinin, somatostatin, and corticotrophin) (12,13). In each of these examples, channel inhibition depends on G␤␥ activity, yet an understanding of the molecular events required for channel inhibition remains incomplete. For instance, dopamine inhibits nickel-sensitive LVA calcium channel currents in sympathetic and dorsal root neurons (14,15), lactrotrophs (16,17), melanotrophs (18), retinal horizontal cells (19), and adrenal zona glomerulosa (ZG) cells (20,21). Yet, as typified in ZG cells, G␤␥ alone is not sufficient to mediate channel inhibition transduced by dopamine, and conflicting data posits the singular importance of activating D 1 -or D 2 -receptor subtypes that produce opposing changes in the level of the intracellular messenger cAMP (20,21).
Using a heterologous expression system, we recently described a molecular mechanism for the inhibition of LVA channels that involves heterotrimeric G-protein ␤␥ dimers (22). Unlike inhibition of HVA channels, inhibition of LVA channel activity by G␤␥ dimers is independent of voltage, specific for Ca v 3.2/␣ 1H channels within the LVA channel family, and requires active G␤␥ dimers that contain G␤ 2 . Yet, like G␤␥-induced HVA channel inhibition, LVA channel inhibition depends on a direct interaction of G␤␥ dimers with the channel protein. Accordingly, recombinant G␤ 2 ␥ 2 dimers inhibit Ca v 3.2 single channel activity when applied to membrane patches excised from HEK293 cells (23). Here, we test the possibility that the described molecular mechanism for the inhibition of Ca v 3.2 channels underlies dopamine-induced inhibition of Ca v 3.2 currents. Our results reconcile previous findings yet are unexpected. They demonstrate that: 1) protein kinase A (PKA) acts as a molecular switch to enable the inhibition of Ca v 3.2 currents by G␤ 2 ␥ 2 dimers, and 2) they highlight a role for a novel cooperative action of D 1 and D 2 receptors in controlling Ca v 3.2 calcium channel activity.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-H295R cells (human adrenocortical carcinoma cells) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% cosmic calf serum, 1 g/ml gentamicin. All culture vessels for H295R cells were coated with sterile 0.1% gelatin and washed with 1ϫ (phosphate-buffered saline) before use. HEK293 cells (human embryonic kidney cells) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 1% pen/strep. Cells were transiently transfected with plasmids for wild-type or mutant Ca v 3.2/Ca v 3.1 channels by using JetPEI reagent (Polyplustransfection.com) and used for patching or immunoprecipitation 48 h after transfection.
Electrophysiology-Ca v 3.2 currents were recorded with an Axopatch 200A amplifier (Axon Instruments), and data collected with PCLAMP 9.2 software (Axon Instruments). All experiments were performed at room temperature. Data were analyzed by using Clampfit 9.2 (Axon Instruments) and Origin-Pro 7.5 (OriginLab) software. ANOVA was used for statistical examination with post hoc Dunnett's test, where significance was taken as p Ͻ 0.05. Average data are given as mean Ϯ S.E.
Whole Cell Recording-Steady-state channel activation was determined using tail currents in response to test depolarization in 5-mV increments (Ϫ60 mV to ϩ10 mV) from a holding potential of Ϫ90 mV upon repolarization to Ϫ60 mV; steadystate channel inactivation was determined in response to ϩ20 mV test depolarization (6 ms) from holding a potential incremented in 5 mV (Ϫ90 mV to Ϫ20 mV; 6 s) upon repolarization to Ϫ60 mV. To follow the time course of agonist-induced current inhibition, repetitive tail currents elicited upon repolarization to Ϫ75 mV from a test pulse of Ϫ15 mV were delivered every 6 s. The internal solution (in mM): 115 CsCl, 1 TBACl, 1 MgCl 2 , 5 Mg-ATP, 1 Li-GTP, 0.9 CaCl 2 , 20 HEPES, 11 BAPTA, pH 7.2 (adjusted with CsOH). The bath solution (in mM): 132 TEACl, 10 CaCl 2 , 0.5 MgCl 2 , 10 HEPES, 5 dextrose, 32 sucrose, pH 7.4 (adjusted with CsOH). Currents were filtered at 2 kHz and sampled at 12.5 kHz, and leak subtraction was performed on line by using scaled hyperpolarizing steps of one-fourth amplitude (P/N4) (22).
Single Channel Recording-Unitary currents from inside-out excised patches were elicited by a test pulse to Ϫ35 mV from Ϫ90 mV (200 ms, 6 s. interpulse). Currents were sampled at 100 kHz and filtered at 2 kHz. The pipette solution (in mM): 75 CsCl, 60 CaCl 2 , and 10 HEPES, pH 7.4 (adjusted with CsOH). The bath solution (in mM): 140 K ϩ -aspartate, 5 MgCl 2 , 10 EGTA, 20 HEPES, pH 7.4 (adjusted with KOH), 0.162 CHAPS (0.01%), and 0.04 dithiothreitol. CHAPS and dithiothreitol were added to the bath solution to maintain the stability and solubility of G␤␥ recombinant proteins. Purified G␤␥ subunits were added directly to the bath solution. Channel openings were detected with a 50% threshold crossing criterion. Open probability (NP o ) was calculated as the ratio of the sum of channel open time (per sweep) and the analyzed test pulse duration (23,24).
Molecular Biology-Ca v 3.2/Ca v 3.1 II-III loop channel chimera was made as reported (22). Mutations of putative PKA phosphorylation sites were introduced by using the Quik-Change XL Site-directed Mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by sequencing.
Generation of Recombinant G␤ x ␥ 2 -The recombinant G␤␥ proteins were made and purified as reported (23). The activity of pure G␤␥ dimer was verified by testing for PLC-␤ activation in synthetic lipid vesicles (25).
Phosphorylation Assay-Recombinant GST fusion proteins of Ca v 3.2 II-III loop and deletion mutants were purified as previously described (26). Each phosphorylation reaction (

PKA Activity Enables the Inhibition of Ca v 3.2 Unitary
Currents by G␤ 2 ␥ 2 -In patches excised from HEK293 cells stably expressing Ca v 3.2 channels, recombinant G␤ 2 ␥ 2 subunits reduce the frequency of Ca v 3.2 unitary openings (23). Here, we confirm these findings in HEK293 cells that are transiently expressing recombinant Ca v 3.2 channels. Calcium current was elicited from a holding potential of Ϫ90 mV by a depolarizing test pulse to Ϫ35 mV. Following patch excision, control activity was measured for 5-6 min during bath perfusion with vehicle, before the direct application of G␤␥ subunits. At Ϫ35 mV, active calcium channels displayed varied patterns of small openings. As illustrated in Fig. 1A from an exemplar recording of an excised patch that may have more than one active channel, channel openings were abundant early in the record producing a transient ensemble averaged current consistent with time-dependent inactivation. G␤ 2 ␥ 2 (1-2 nM) reduced (41%) the channel open probability (NPo) per sweep, a value calculated from the ratio of the total open time and the test pulse duration, and increased (193%) the number of silent/null sweeps (denoted by the solid circles). Surprisingly, these findings were not replicated in human adrenocortical carcinoma cells (NCI-H295R) transiently expressing recombinant human Ca v 3.2 channels. Rather, we observed that G␤ 2 ␥ 2 failed to inhibit Ca v 3.2 single channel currents recorded from patches excised from unstimu-lated NCI-H295R cells; neither the single channel probability (NPo) per sweep, nor the number of silent sweeps was changed by the application of G␤ 2 ␥ 2 (Fig. 1B). To reconcile these findings, we considered the possibility that activation of an additional second messenger system that remained quiescent in unstimulated H295R cells was required for current inhibition.
A role for PKA in the regulation of LVA current is supported by numerous electrophysiological studies; however, the resulting alteration in channel activity seems context dependent and complex. PKA activity increases recombinant currents expressed in Xenopus oocytes (27), mammalian cells (HEK293 or CHO) (28), and bullfrog atrial myocytes (29) yet cAMP analogues do not increase native mammalian LVA currents (16, 20, 30 -32). Nevertheless, pharmacologic inhibition of PKA activity can prevent the stimulation (33,34) or even the inhibition (19,20,35) of native LVA currents induced by G-protein receptor activation. Accordingly, we considered the possibility that PKA mediated phosphorylation of the channel protein may be required for G␤ 2 ␥ 2 -mediated inhibition of Ca v 3.2 single channel currents. We treated H295R cells expressing Ca v 3.2 channels with 10 M 8-Br-cAMP for 10 min prior to patch excision, and tested for inhibition. G␤ 2 ␥ 2 markedly decreased NPo (diary plot, Fig. 1D) and concomitantly increased the number of silent sweeps. The G␤ 2 ␥ 2 -mediated decrease in sweep NPo averaged 35 Ϯ 3% (n ϭ 6) and was accompanied by a 131 Ϯ 44% increase in the number of null sweeps. These changes in channel open probability were not elicited by G␤ 1 ␥ 2 dimers (Fig. 1E) and were not complemented by changes in: the first latency to open (50.7 Ϯ 4.9 ms (cAMP), 50.4 Ϯ 0.6 ms (cAMP ϩ ␤ 2 ␥ 2 ), the single channel conductance of (6.6 pS with 60 mM CaCl 2 ), or the open-state distributions of dwell times, which remained fitted to the sum of two exponentials with unchanged proportions (cAMP ϩ ␤ 2 ␥ 2 : 1 ϭ 0.60 Ϯ 0.14 ms (62%), 2 ϭ 2.61Ϯ.26 ms Numbers are patches recorded for each condition. *, p Ͻ 0.05 compares vehicle with treatment group by t test. B, unitary currents recorded from wild-type Ca v 3.2 channels expressed in H295R cells without or (C) with 10 M 8-Br-cAMP pretreatment (10 min, room temperature) before patch excision. D, diary plot shows the time course of NPo from the representative patch (without, left; with, right) 10 M 8-Br-cAMP pretreatment. E, bar graph plots mean Ϯ S.E. of percent reduction in NPo (left) or percent increase in nulls (right) elicited by G␤ 2 ␥ 2 or G␤ 1 ␥ 2 relative to vehicle exposure for each patch. Numbers are patches recorded for each condition. *, p Ͻ 0.05 compares vehicle with treatment groups by ANOVA.
Dopamine Induces a Voltage-independent Inhibition of Ca v 3.2 Current That Requires Active G␤␥ Dimers and PKA-Dopamine mediates its actions via two distinct classes of receptors: D 1 -like (D 1 , D 5 ) that couple to the heterotrimeric G protein G␣ s , and D 2 -like receptors (D 2 , D 3 , D 4 ) that couple to G␣ i / G␣ o (37). D 1 -, and D 2 -like receptors (D 2l/ D 2s and D 4 ) receptors are expressed in the human and rodent adrenal cortex, and in human NCI-H295R adrenocortical carcinoma cells (38,39). Because dopamine inhibits nickel-sensitive LVA currents in many native cells including the aldosterone producing ZG cell, we characterized inhibition of recombinant Ca v 3.2 channels transduced by endogenous dopamine receptors in H295R cells that lacked native Ca v 3.2 channel expression. Dopamine produced current inhibition (Fig. 2, A and B) that was slow in onset ( Fig. 2A), depended on dose (1-50 M; Fig. 2B), occurred at all test potentials (Ϫ45 to ϩ15 mV; data not shown), and was not accompanied by either a change in the voltage-dependence of activation (Fig. 2C) or inactivation (Fig. 2D). Therefore, this scaled reduction in Ca v 3.2 whole cell calcium current in H295R cells by dopamine mimics the voltage-independent inhibition of current induced by G␤ 2 ␥ 2 dimers in HEK293 cells (22,23) expressing human recombinant Ca v 3.2 channels. Therefore, we perturbed dopamine signaling to determine whether cAMP and active G␤␥ subunits are required for inhibition of Ca v 3.2 current by dopamine. We provided a "sink" for active G␤␥ by transfecting H295R cells with either transducin, the sensory rhodopsin II G␣ subunit, or ␤ARKct, the C terminus of ␤-adrenergic receptor kinase-1 (40). The expression of either transducin or ␤ARKct prevented the inhibition of Ca v 3.2 current induced by dopamine (Fig. 3A). Current levels remained indistinguishable from those of vehicle-treated cells transfected with empty vector (vehicle: 1.1 Ϯ 1.5%, n ϭ 8; ␤␥ sinks combined: 6 Ϯ 2.4%, n ϭ 9). Because PKA plays a central role in dopamine signaling, and 8-Br-derivatives of cyclic nucleotides preferentially target nucleotide activated protein kinases (41), we evaluated PKA as the cAMP effector molecule. To prevent PKA activation by dopamine, we used PKI (5-24), a selective peptide inhibitor that binds to the catalytic subunit of PKA and is a more specific inhibitor of PKA than either H89 or KT5720 (42) (Fig. 3B). Inclusion of PKI in the patch pipette precluded channel inhibition induced by dopamine but failed to inhibit Ca v 3.2 channel current in vehicle-treated cells (vehicle: 2.7 Ϯ 1.5%, n ϭ 11; PKI: 0.4 Ϯ 2.3%, n ϭ 5; n.s.). Collectively, these data identify PKA as the cAMP effector and provide support for a cooperative role for active G␤␥ subunits and PKA in the regulation of channel activity by dopamine.

II-III Intracellular Loop Harbors a Critical PKA Phosphorylation Site for Channel
Regulation-Inhibition of Ca v 3.2 channels by heterologously expressed G␤ 2 ␥ 2 dimers depends on the intracellular loop that connects transmembrane domains II and III. Replacing the II-III loop of Ca v 3.2 channels with that of unregulated Ca v 3.1 channels prevents current inhibition (22). To test the importance of the II-III loop in the regulation of LVA channels by dopamine, we evaluated the regulation of a chimeric Ca v 3.2 channel (Ca v 3.2 (Ca v 3.1 II-III) that contains the II-III loop from Ca v 3.1 channels. Each of three channel constructs: wild-type Ca v 3.1, wild-type Ca v 3.2, or chimeric Ca v 3.2, Ca v 3.2(Ca v 3.1 II-III) was transiently expressed in H295R cells and tested for dopamine-induced current inhibition (Fig. 4A). Dopamine reduced Ca v 3.2 channel current by 21.3 Ϯ 1.6% (n ϭ 15), but failed to decrease current carried by Ca v 3.1 channels (4.0 Ϯ 3.0%, n ϭ 9). Ca v 3.2 chimeric channels also were refractory to inhibition by dopamine (4.5 Ϯ 3.2%, n ϭ 8) despite displaying gating properties similar to wild-type Ca v 3.2 channels. These data are in agreement with channel subtype-specific regulation by G␤␥ subunits, and the important role of the II-III loop in this mechanism of regulation.
The Ca v 3.2 channel protein contains 25 phosphorylation sites that conform to the minimal consensus sequence for PKA phosphorylation (R/R/X/(S/T)/X-hydrophobic). We evaluated the highest stringency PKA site in the channel protein located on the C terminus (Arg-Arg-Arg-Thr 2214 -Pro) and the two highest stringency sites located on the II-III loop (Arg-Arg-Ser-Ser 1144 -Trp; Arg-Arg-Gly-Ser 1107 -Ser), as potential sites for PKA regulation (Fig. 4A). These selected residues in the full-length channel protein were mutated alone or in combination, and currents carried by the expressed channel constructs were tested in H295R cells for agonist-induced inhibition. The combined mutation of residues: S1144A, S1107A, and T2214A, produced a triple mutant channel that had normal gating properties (data not shown). Notably, this mutant channel was refractory to modulation by dopamine. Following agonist exposure, current levels remained indistinguishable from those of vehicle-treated cells expressing wild-type Ca v 3.2 channels (% inhibition: vehicle: 1.4 Ϯ 1.5%, n ϭ 17; triple mutant: 5.1 Ϯ 1.5%, n ϭ 7; n.s.). Channels harboring the Ser 1107 mutation were similarly refractory to inhibition by dopamine (4.9 Ϯ 3.9%, n ϭ 8; n.s.). This inhibition of modulation was specific for the Ser 1107 site, as calcium current carried by Ca v 3.2 channels harboring either an adjacent mutation in the II-III loop (Ser 1144 ) or one in the channel C terminus (Thr 2214 , data not shown) were regulated by dopamine. To confirm that the Arg-Arg-Gly-Ser 1107 -Ser recognition motif on the II-III loop is indeed a site for PKA phosphorylation, we used GST fusion proteins that expressed either full-length or abbreviated II-III loop sequences that internally removed one or both of the PKA recognition motifs (ϳ60 amino acids) as substrates in in vitro phosphorylation reactions. The catalytic subunit of PKA robustly phosphorylated the full-length loop (amino acids 1019 -1300) and the abbreviated loop devoid of the S1144 recognition motif (d: 1142-1192) to nearly equal extent (Fig. 4B). By contrast, removal of the S1107 recognition motif alone (d:1059 -1108) or in combination with that of S1144 (d:1097-1154) dramatically decreased the level of incorporated phosphate (Fig. 4B). Moreover, because deletion of the S1144 recognition motif reduced phosphate incorporation only when the S1107 motif was also removed, our data imply that Ser 1107 is the preferred phosphorylation site in the full-length II-III loop. Collectively our mutagenesis studies highlight the critical role played by PKA-mediated phosphorylation and identify the phosphorylation state of residue Ser 1107 as a molecular switch for the control of Ca v 3.2 channel activity induced by dopamine.
Based on these findings, we hypothesized that the efficacy of G␤ 2 ␥ 2 in the excised-patch also would be dependent on the phosphorylation state of Ser 1107 . Accordingly, G␤ 2 ␥ 2 would not be expected to inhibit Ca v 3.2 single channel currents carried by channels harboring the S1107A mutation. As predicted, S1107A mutant Ca v 3.2 channels were refractory to modulation by G␤ 2 ␥ 2 (Fig. 5, A-C) as they were refractory to modulation by dopamine. Importantly, this dependence on the phosphorylation state of Ser 1107 for G␤ 2 ␥ 2 inhibitory activity was not a specific requirement of H295R cells. S1107A mutant Ca v 3.2 channels expressed in HEK293 cells were also refractory to modulation by G␤ 2 ␥ 2 in the excised-patch (Fig. 5, A-C). Hence the high cAMP tone of HEK293 cells (43), likely promotes PKA activation and precludes the need for cellular pretreatment with cAMP, permitting the regulation of wild-type Ca v 3.2 channels by G␤ 2 ␥ 2 subunits. To provide additional experimental support for this rationale, we analyzed the state of phosphorylation of FLAG-tagged Ca v 3.2 channels expressed in HEK293 cells using a phospho-(Ser/Thr) PKA substrate antibody. Wild-type, single (Ser 1107 ), and triple (S1144A, S1107A, and T2214A) mutant channels expressed in HEK293 cells were immunoprecipitated, resolved by SDS-PAGE, and immunoblotted with phospho-(Ser/Thr) PKA substrate antibody. As illustrated in supplemental Fig. S2, wild-type Ca v 3.2 channels demonstrated a high degree of phosphorylation, which in part, was attributable to PKA activity as the states of phosphorylation of the PKA-triple and the single S1107A mutant channel were proportionally reduced.
We questioned further whether phosphorylation of the channel by PKA was required for binding or inhibitory action of G␤ 2 ␥ 2 subunits. We immunoprecipitated cAMP-phosphorylated Ca v 3.2 channels from HEK293 cells and tested binding of recombinant G␤␥ dimers to the channel protein in vitro. After extensive washing, bound G␤␥ dimers were separated from FLAG-tagged channels by SDS-PAGE and detected in immunoblots using ␤-specific antibodies. Consistent with selective modulation of Ca v 3.2 channels by G␤ 2 -containing dimers, recombinant G␤ 2 ␥ 2 but not G␤ 1 ␥ 2 dimers, bound to the immunoprecipitated channel protein (Fig. 6A). The measured binding was specific to the channel immunopre-cipitate as binding to an immunoglobulin immunoprecipitate was null. Importantly, binding of G␤ 2 ␥ 2 dimers to S1107A mutant Ca v 3.2 channels was equivalent to that of wild-type channels implying that phosphorylation of Ser 1107 , in addition to binding, is needed for G␤ 2 ␥ 2 inhibitory activity (Fig. 6B). Collectively these data identify the mechanism underlying regulation of Ca v 3.2 channels by G␤ 2 ␥ 2 dimers and show that phosphorylation of Ser 1107 acts as a molecular switch to enable G␤ 2 ␥ 2 inhibitory activity.

Co-activation of D 1 and D 2 Receptors Is Required for Channel
Inhibition-The relative contribution of D 1 versus D 2 receptors to current inhibition in ZG cells is ambiguous with data supporting roles for each receptor subtype (20,21). Typically, D 1 and D 2 receptors transduce opposing alterations in cAMP signaling that underlies their antagonistic actions. However, G i/olinked D 2 receptors can contribute to the activation of PKA (44) if cAMP is generated by specific adenylyl cyclase subtypes (AC2, AC4, AC7). These AC subtypes are resistant to inhibition by G␣ i subunits , and are activated by G␤␥ dimers released from G i/o -linked receptors (45). Notably, AC subtypes 2 and 3 are expressed robustly in the plasma membrane compartment of the human adrenal (39,46), and in ZG cells activation of G␣ i -linked D 2 -receptors does not reduce cAMP formation produced by active G␣ s (37). Therefore, we considered the possibility that D 1 and D 2 receptors may cooperate to mediate dopamine-induced current inhibition. We used D 1 or D 2 receptor antagonists to block receptor activation and evaluated regulation of channel activity by dopamine. Preexposure to either a D 1 -selective (SCH-23990) or a D 2 -selective (raclopride) antagonist prevented inhibition of channel current by dopamine (Fig. 7A, dopamine: 23.1 Ϯ 2.4%, n ϭ 9; ϩ10 M SCH-23990: 7.6 Ϯ 2.7%, n ϭ 7; ϩ20 M raclopride: 6.4 Ϯ 1.7%, n ϭ 6; p Ͻ 0.05 versus DA without antagonists). In addition we measured current inhibition transduced by either D 1 (SKF-38393) or D 2 (bromocriptine) receptor agonists used alone or in combination. Each agonist transduced a dose-dependent inhibition of channel current attaining a maximal level of current inhibition equivalent to that induced by dopamine. However in support of receptor coactivation, each agonist alone failed to modify channel activity when used at a dose that was receptor-selective (1 1 II-III) channel chimera, and the locations of PKA phosphorylation sites selected for mutation (S1107A, S1144A, T2214A). Middle panel, histogram plots mean Ϯ S.E. of percent inhibition of current density recorded from H295R cells 10 min after the application of 50 M dopamine. Note, S1107 is permissive for dopamine-induced current inhibition. Numbers are cells patched for each condition. *, p Ͻ 0.05 compares control with treatment group by ANOVA. Right panel, sample currents recorded at Ϫ15 mV from holding Ϫ90 mV following repolarization to Ϫ75 mV for each condition. B, in vitro phosphorylation of full-length or abbreviated Ca v 3.2 II-III loop GST fusion proteins by the catalytic unit of PKA. Numbers denote residue positions in the full-length channel. WT, full-length II-III loop, amino acids 1019 -1300; ϪS1107, amino acids 1059 -1107 deleted; ϪS1144: amino acids 1142-1192 deleted; ϪS1107/ϪS114: amino acids 1097-1154 deleted removing PKA phosphorylation recognition motifs alone or in combination. Coomassie Blue detection of GST proteins show equivalent protein loading. Notably S1107 is the preferred PKA phosphorylation motif on the full-length loop.
M) (37), but when added together synergized to inhibit Ca v 3.2 channel currents (10.2 Ϯ 0.5%, n ϭ 5). By contrast, at 2 M each agonist alone failed to inhibit channel activity. Taken together, these data indicate that coactivation of D 1 and D 2 receptors is required for channel inhibition.
Based on the mechanism described above for channel inhibition that requires the dual activity of PKA and released G␤ 2 ␥ x dimers, we examined further the contribution of G i/o -linked D 2 receptors and G s -linked D 1 receptors to second messenger generation. We hypothesized that if active D 2 receptors preferentially released G␤ 2 ␥ x subunits, and D 1 -receptors preferentially generated cAMP, (Fig. 7D), we would expect cAMP treatment to permit the inhibition of Ca v 3.2 channel activity by D 2 -recep-tor agonists but not D 1 -receptor agonists. 10 M 8-Br-cAMP was ineffective alone (see Fig. 1A) and failed to facilitate current inhibition produced by the D 1 receptor agonist SKF (1 M), but enabled the inhibition of current produced by the D 2 receptor agonist bromocriptine (1 M, Fig. 7C). Moreover, this current inhibition induced by bromocriptine was prevented by preexposure to the D 2 receptor antagonist, raclopride (20 M) but not by the D 1 receptor antagonist, SCH-23390 (10 M). Thus, our data provide support for an underlying intracellular mechanism whereby each dopamine receptor subtype preferentially contributes one critical signal toward producing the inhibition of Ca v 3.2 channel activity.

DISCUSSION
Our results define a molecular mechanism by which PKA controls inhibition of Ca v 3.2 channels by G␤␥ dimers and offer a cellular explanation for the regulation of LVA calcium channels by dopamine. These data describe how cAMP combines with active G␤␥ dimers to inhibit Ca v 3.2 channels and unites previous observations that have suggested the importance of these signaling molecules in dopamine-induced current inhibition (20). We show that PKA is the effector protein for cAMP

channels in vitro.
Immunoprecipitation of FLAG-tagged Ca v 3.2 channels from HEK293 cell lysates using a Ca v 3.2 channel specific antibody or a goat IgG. A, binding of 10 nmol of recombinant G␤ x ␥ 2 dimer: G␤ 2 ␥ 2 (left), or G␤ 1 ␥ 2 (right) to the immunoprecipitate. Immunoblots show bound G␤ subunits detected with G␤-specific antibodies after resolution on SDS-PAGE. B, binding of G␤ 2 ␥ 2 dimers to wild-type and S1107A mutant channel proteins was compared. FLAG-tagged Ca v 3.2 channel protein in immunoprecipitates was detected with Anti-FLAG antibody. Shown is representative experiment (n ϭ 4). Notably, S1107 was not required for G␤ 2 ␥ 2 channel specific binding. and that PKA activity acts as a molecular switch to permit the inhibition of Ca v 3.2 channels by G␤ 2 ␥ 2 subunits.
The phosphorylation state of a critical serine residue (Ser 1107 ) located on the II-III loop of the channel protein governs the regulation of channel activity by G␤␥ subunits. Neither the phosphorylation state of an adjacent PKA site (Ser 1144 ) on the II-III loop with equivalent stringency nor the phosphorylation state of the highest stringency site on the channel protein (Thr 2214 ) is permissive for channel regulation by G␤␥ subunits. Significantly, this requirement for channel phosphorylation provides new possibilities for precise tuning of calcium signaling via the control of cyclic nucleotide phosphodiesterase and/or protein phosphatase activities. G␤ 2 -containing ␤␥ dimers are the only dimeric isoforms that bind to the channel II-III loop and reduce the open probability of Ca v 3.2 channels (22,23). Here we show that PKA activity does not change G␤ subtype selectivity but rather allows G␤ 2 ␥ 2 dimers to silence channels in the membrane, decreasing their frequency of opening. This requirement for channel phosphorylation is not cell context dependent as our data show that Ca v 3.2 channels expressed in HEK293 cells are basally phosphorylated and recombinant G␤ 2 ␥ 2 dimers do not regulate unitary currents carried by S1107A mutant channels expressed in either H295R or HEK293 cells. Ser 1107 phosphorylation does not regulate G␤ 2 ␥ 2 binding to the channel protein; hence we conclude that the phosphorylation of Ser 1107 in addition to G␤ 2 ␥ 2 binding is required to inhibit Ca v 3.2 channels. Interestingly, phosphorylation of Ser 1198 in the II-III loop is critical for CaMKII regulation of Ca v 3.2 channel activity (47) suggesting an important role for II-III loop phosphorylation in Ca v 3.2 channel gating.
Although D 1 /D 2 receptor coactivation typically results in antagonism at the cellular level (37,48), our studies provide evidence for a novel cellular mechanism by which D 1 /D 2 receptors coordinate to produce a cellular response that differs from other receptor coactivation paradigms previously reported that have underscored the production of either an amplified signal (44) or the generation of a novel second messenger that is not produced by activation of either receptor alone (32). Here, we show that each receptor independently produces a signal (cAMP/active G␤ 2 ␥ x subunits) that is required to mediate current inhibition. Notably, although our studies do not rule out the possibility that activation of G␤ 2 ␥ x subunits by G i/o -linked D 2 receptors may contribute to cAMP formation by stimulating G␤␥-sensitive adenylyl cyclase isoforms, our data indicate that cAMP-independent signaling also is required to elicit current inhibition, as the application of 8Br-cAMP alone failed to reduce either Ca v 3.2 whole-cell or single channel current. Thus, the signaling paradigm outlined here differs from that described in the nucleus accumbens where coactivation of D 1 and D 2 receptors coordinate to increase spike firing by amplifying the production of a single second messenger, cAMP (44). Dual modulation of T-type calcium channels by PKA and G i /G o proteins also underlies amplified facilitation of LVA currents in frog atrial myocytes although the intracellular mechanism of this potentiation remains undefined (29).
In addition, our data highlight the previously underappreciated role of receptor co-activation in dopamine induced LVA current inhibition. Our proposed dual receptor mechanism is consistent with previously reported studies that either have focused solely on the effectiveness of D 1 or D 2 -antagonism in preventing dopamine-induced current inhibition (19 -21, 49); or have underscored the ineffectiveness of D 1 -antagonism by circumventing D 1 receptor signaling with intracellular cAMP fixed at high levels (17). Nevertheless, in pituitary melanotrophs D 2 agonists used at receptor selective concentrations induce current inhibition without apparent D 1 receptor co-ac- tivation (18). Because the most sensitive inhibitory current responses to dopamine have been reported in pituitary lactotrophs when cAMP is fixed at 200 M (17), and because 10 M 8-Br-cAMP potentiates current inhibition by dopamine in H295R cells, 4 we speculate that differences in the expression of G␤␥-sensitive adenylyl cyclase isoforms and hence PKA activity may obviate a requirement for D 1 receptor activation in some cellular preparations.
Collectively our data show that protein kinase A acts as a molecular switch to enable the voltage-independent inhibition of Ca v 3.2 currents by G␤ 2 ␥ x subunits and provides a mechanism for expanding the dynamic range of Ca v 3.2 channel activity. In the adrenal glomerulosa cell, where aldosterone production is driven by Ca v 3.2 channel activity, failure of this mechanism may contribute to aldosterone excess and the loss of inhibitory dopaminergic tone that is observed in some forms of human hypertension (50 -54).