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CELLULAR AND MOLECULAR

Augmentation of Ca_v3.2 T-Type Calcium Channel Activity by cAMP-Dependent Protein Kinase A

Department of Life Science (J.-A.K., J.-Y.P., H.-W.K., S.-U.H., J.-H.L.) and Interdisciplinary Program of Integrated Biotechnology (J.-A.K., J.-H.L.), Sogang University, Seoul, Korea; and Department of Physiology, Institute of Basic Medical Science, Yonsei University Wonju College of Medicine, Ilsan-Dong Wonju, Korea (S.-W.J.)

Received January 14, 2006; accepted March 27, 2006.

	Abstract

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Ca²⁺ influx through T-type Ca²⁺ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Ca_v3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 µM forskolin, an adenylyl cyclase stimulant, increased Ca_v3.2 channel activity by 40 ± 4% over 30 min and negatively shifted the steady-state inactivation curve (V₅₀ = -61.4 ± 0.2 versus -65.5 ± 0.1 mV). Forskolin did not affect other biophysical properties of Ca_v3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 µM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Ca_v3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 µM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Ca_v3.2 channel activity by PKA was mimicked by serotonin when 5HT₇ receptor was coexpressed with Ca_v3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Ca_v3.2 with those of the Na_v1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Ca_v3.2.

To date, molecular biological studies have identified 10 genes encoding the voltage-activated calcium channel ₁ subunits that determine the primary biophysical and pharmacological properties of the channels. Expression studies have revealed that the Ca_v3.1 (_1G), Ca_v3.2 (_1H), and Ca_v3.3 (_1I) genes encode low-voltage-activated T-type Ca²⁺ channel ₁ subunits, whereas the other genes encode high-voltage-activated Ca²⁺ channel ₁ subunits. The three T-type Ca²⁺ channels have been produced in expression systems and have been shown to possess the following biophysical and pharmacological properties: 1) activation thresholds around a resting membrane potential of -60 to -70 mV, 2) inactivation at low voltages, 3) slow deactivation, 4) tiny single channel conductance, and 5) high sensitivity to kurtoxin and mibefradil. The Ca_v3.1 and Ca_v3.2 channels generate typical T-type channel currents with transient kinetics because of fast activation and subsequent inactivation. In contrast, Ca_v3.3 channels produce atypical T-type channel currents with much slower kinetics.

T-type channels are primarily regulated by dynamic changes in membrane potential. Numerous studies have revealed that they are also affected by hormones and/or neurotransmitters via Ca²⁺/calmodulin-dependent protein kinase II (Welsby et al., 2003) and protein kinase C (Park et al., 2003). Interestingly, G protein subunits have shown to make a negative regulation effect on Ca_v3.2 T-type channel activity by interacting with the cytoplasmic loop connecting domain II and III (Wolfe et al., 2003). It has recently shown that treatment of cAMP or -adrenergic stimulation could increase T-type channel activity in rat chromaffin cells through Epac-dependent recruitment of T-type channels (Novara et al., 2004; Giancippoli et al., 2006).

There is controversy about whether they are regulated by cAMP-dependent protein kinase A (PKA). The majority of investigators have reported that T-type channels are little affected by PKA (Bean, 1985; Benham and Tsien, 1988; Hagiwara et al., 1988; Tytgat et al., 1988; Hirano et al., 1989; Tseng and Boyden, 1989; Fisher and Johnston, 1990). In contrast, T-type Ca²⁺ currents in frog atrial myocytes were reported to be increased by isoproterenol in two ways: a cAMP-dependent way and a cAMP-independent way (Alvarez and Vassort, 1992). In addition, Lenglet et al. (2002) also reported that T-type channel activity recorded in rat glomerulosa cells was augmented by PKA after stimulation of 5HT₇ receptors.

In the present investigation, we sought to resolve the question whether T-type channel activity is regulated by PKA, using T-type Ca_v3.2 Ca²⁺ channels reconstituted in the Xenopus oocyte system. We found that Ca_v3.2 channel activity was significantly increased by forskolin-activated PKA. This PKA effect could be mimicked by serotonin when 5HT₇ receptor was coexpressed with Ca_v3.2 in Xenopus oocytes. In addition, we localized the region(s) of the Ca_v3.2 channel responsible for PKA stimulation to the cytoplasmic loop connecting domains II and III.

	Materials and Methods

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Materials
Forskolin, H89, 5-hydroxytryptamine (5-HT; serotonin), 8-bromo (Br)-cAMP, 8-bromoadenosine-3',5'-cyclic monophosphorothioate (RpBrcAMPs), and protein kinase A inhibitor peptide (PKI) were purchased from Sigma-Aldrich (St. Louis, MO). Forskolin and H89 were diluted in dimethyl sulfoxide to generate 10 mM stock solutions. The concentration of dimethyl sulfoxide in the bath solution is expected to be less than 0.1%, which had no effect on T-type currents. PKI, 8-Br-cAMP, and RpBrcAMPs were prepared at stock concentrations of 1, 100, and 100 mM in double distilled water, respectively.

Construction of Chimeric Channels and the Deletion Mutant
The chimeric channels Ca_v3.2/Na_v1.4_N-term, Ca_v3.2/Na_v1.4_I-II, Ca_v3.2/Na_v1.4_II-III, and Ca_v3.2/Na_v1.4_III-IV were created by replacing individual cytoplasmic loops encoded by human Ca_v3.2 cDNA (_1H; GenBank accession number AF051946 [GenBank] ) with the corresponding loops of rat Na_v1.4 cDNA (µ-1; GenBank accession number NM_013178 [GenBank] ) by overlap extension PCR (Horton et al., 1989). A rat 5HT₇ receptor cDNA was obtained by reverse transcription-PCR from adrenal gland total RNA from Sprague-Dawley rats. All PCRs were performed using Pfu DNA polymerase (Genaxxon BioScience, Biberach, Germany). PCR products were inserted into TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced. Error-free PCR products were subcloned into the original Ca_v3.2 pGEM-HEA using restriction enzyme sites (Chuang et al., 1998). Detailed information about the construction of the individual chimeric channels is given below.

Ca_v3.2/Na_v1.4_N-term. The N terminus (5' polylinker-849) of Na_v1.4 was amplified from rat Na_v1.4 cDNA using forward primer 5'-TAATACGACTCACTATAGGG-3' (T7 primer) and reverse primer 5'-CACGTGCTCGAACCATGGGAACAGCGCGTGAATGAG-3'. By overlap extension PCR, the amplified N-terminal cDNA was connected to the domain I portion (377-1227) of Ca_v3.2 amplified with forward primer 5'-CTCATTCACGCGCTGTTCCCATGGTTCGAGCACGTG-3' and reverse primer 5'-GATGTCGACCCAGCCTTCCAG-3', respectively. The extended cDNA was digested with ClaI (5'-polylinker) and BamHI (729, Ca_v3.2) and ligated into Ca_v3.2 pGEM-HEA opened with ClaI (5'-polylinker) and BamHI (729, Ca_v3.2).

Ca_v3.2/Na_v1.4_I-II. The cytoplasmic I-II loop (1360-2434) of Ca_v3.2 was replaced with the corresponding loop (1782-2162) of Na_v1.4. The I-II loop (1782-2162) of Na_v1.4 was amplified with forward primer 5'-TTCTCGGAGACGAAGCAGGCTGAGCAGAATGAGGC T-3' and reverse primer 5'-GTCCACGATGCGGCGCAGGTCCATGACGATCAGGTA-3'. The upstream portion (nucleotides 305-1360) preceding the Ca_v3.2 I-II loop was amplified with forward primer 5'-GCGGCCACGGTCTTCTTCTG-3' and reverse primer 5'-AGCCTCATTC TGCTCAGCCTGCTTCGTCTCCGAGAA-3'. The downstream portion (2435-3116) of the Ca_v3.2 I-II loop was amplified using the forward primer 5'-TACCTGATCGTCATGGACCT GCGCCGCATCGTGGAC-3' and reverse primer 5'-TGGCCACCAGCAGGTTGAAG-3'. The three PCR products were joined together by overlap extension PCR, and the extended cDNA was digested with NotI and BspEI and ligated into Ca_v3.2 pGEM-HEA, which was opened with NotI (341) and BspEI (2637).

Ca_v3.2/Na_v1.4_II-III. The II-III loop (3134-3958) of Ca_v3.2 was replaced with the corresponding loop (2847-3518) of Na_v1.4. The II-III loop (2847-3518) of Na_v1.4 was amplified from Na_v1.4 cDNA using forward primer 5'-ATCCTCGTGGAGGGCTTC AGTGCTGACAGCCTGGCG-3' and reverse primer 5'-CACGTGATCAAACATCTT GTGCTCAACAATCTTGAA-3'. The upstream portion (2435-3133) preceding the Ca_v3.2 II-III loop was amplified using forward primer 5'-CGTCCGGAGCATCGTGGACAGCAA-3' and reverse primer 5'-CGCCAGGCTGTCAGCACTGAAGCCCTCCACGAGGAT-3'. The downstream portion (3959-4401) of the Ca_v3.2 II-III loop was amplified using forward primer 5'-TTCAAGATTGTTGAGCACAAGATGTTTGATCACGTG-3' and reverse primer 5'-AAGAAGGCGCAGCAGATGAG-3'. The II-III loop cDNA and its upstream and downstream cDNAs were joined by further PCR. The extended cDNA was digested with BspEI and EcoRV and ligated into Ca_v3.2 pGEM-HEA that was digested with BspEI (2637, Ca_v3.2) and EcoRV (4350, Ca_v3.2).

Ca_v3.2/Na_v1.4_III-IV. The III-IV loop (4759-4915) of Ca_v3.2 was replaced with the corresponding loop (4329-4485) of Na_v1.4. The III-IV loop (4329-4485) of Na_v1.4 was amplified from rat Na_v1.4 cDNA using forward primer 5'-GTCGAGAACTTCCACAA GCAGAAGAAGAAGTTTGGA-3' and reverse primer 5'-ATAGTGGCTGGTGCACAGC GTCACGAAGTCGTACAC-3'. The preceding portion (3957-4759) of the Ca_v3.2 III-IV loop cDNA was amplified using forward primer 5'-ACAAGCTTTTTGAYCAYGTGGTCCT-3' and reverse primer 5'-TCCAAACTTCTTCTTCTGCTTGTGGAAGTTCTCGAC-3'. The following portion (4916-6196) of the Ca_v3.2 III-IV loop was amplified using forward primer 5'-GTGTACGACTTCGTGACGCTGTGCACCAGCCACTAT-3' and reverse primer 5'-CTCT GCAGGATCCAGGGT-3'. The three amplified fragments were extended by additional PCR. The extended cDNA was digested with EcoRV and AvrII, and ligated into Ca_v3.2 pGEM-HEA, which was opened with EcoRV (4350, Ca_v3.2) and AvrII (6170, Ca_v3.2).

Ca_v3.2_C. Ca_v3.2_C was constructed by deleting the carboxyl-terminal portion (5774-6996) of Ca_v3.2. The portion (3959-5773) preceding the Ca_v3.2 carboxyl terminus was amplified by PCR using forward primer 5'-ACAAGCTTTTTGAYCAYGTGGTCCT-3' and reverse primer 5'-GGGATCCTGTCCGCGTCCA-3'. The PCR product (3959-5774) was digested with SalI and BamHI and ligated into Ca_v3.2 pGEM-HEA digested with SalI (4634) and BamHI (polylinker).

Cloning of Rat 5HT₇ Receptor cDNA
Rat adrenal gland RNA was isolated by the guanidinium thiocyanate-phenol-chloroform extraction method. The first strand cDNA was synthesized from 0.5 µg of rat adrenal gland RNA with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN) by incubation at 22°C for 10 min and then at 42°C for 50 min. The reaction was terminated by heating at 95°C for 5 min. PCR was performed using a pair of PCR primers designed based on the rat 5HT₇ receptor sequence (GenBank access numbers NM_022938 [GenBank] ). The primer sequences were as follows: forward primer 5'-GGCGCTCGGCACGATGATGGA-3' and reverse primer 5'-AGCCAA TGATTTCGTTGTGTTG-3'. The PCR reaction consisted of initial denaturation at 95°C for 1 min, followed by 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min. The resulting PCR products were separated on a 1% agarose gel and purified using a gel extraction column. The purified products were ligated into TOPO TA cloning vector and transformed into competent cells. One of three PCR products sequenced was identical to the open reading frame of NM_022938 [GenBank] . The 5HT₇ receptor cDNA was subcloned into pGEM-HEA.

Expression of the Ca_v3.2 Channel, the Rat Na_v1.4 Channel, Their Chimeric Channels, and the 5HT₇ Receptor in Xenopus Oocytes
Several ovary lobes were surgically isolated from mature female Xenopus laevis (Xenopus Express, Haute-Loire, France) anesthetized with 0.1% of 3-aminobenzoic acid ethyl ester (Sigma-Aldrich). The isolated lobes were manually torn into small clusters of five to six oocytes in SOS solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM pyruvic acid, and 50 µg/ml gentamicin, pH 7.6). Collagenase (type IA, 2 mg/ml; Sigma-Aldrich) dissolved in Ca²⁺-free OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.6) was treated for approximately 30 min to remove the follicle membranes of the isolated oocytes. Stage V to VI oocytes were manually selected under a stereomicroscope and recovered in SOS solution at 18°C for several hours or overnight. Each oocyte was injected with 3 to 10 ng of cRNA in a volume of 40 nl using a Nanojector (Drummond Scientific, Parkway, PA) attached to a micromanipulator (Narishige, Tokyo, Japan). All cDNAs of the T-type channel, the sodium channel, their mutant channels, and 5HT₇ receptor were linearized with AflII or XbaI and transcribed by T7 polymerase using mMESSAGE mMACHINE T7 kits purchased from Ambion (Austin, TX). For the wild-type and chimeric channels (Ca_v3.2/Na_v1.4_N-term, Ca_v3.2/Na_v1.4_I-II, Ca_v3.2/Na_v1.4_II-III, and Ca_v3.2/Na_v1.4_III-IV), 3 to 10 ng of cRNA was injected into oocytes to compare their relative expression levels. There was no significant difference between current amplitudes of the wild-type and chimeric channels. In contrast, the expression level of Ca_v3.2_C was significantly smaller than that of the Ca_v3.2 (P < 0.01, Student's t test). The synthesized cRNA was resuspended in diethyl pyrocarbonate-treated H₂O and stored at -70°C. The 5HT₇ receptor and Ca_v3.2 channel cRNAs were injected in a molar ratio of 1:1.

Electrophysiological Recordings and Data Analysis
Whole-cell currents were measured with a two-microelectrode voltage-clamp amplifier (OC-725C; Warner Instruments, Hamden, CT) between the days 3 and 8 after cRNA injection. Microelectrodes were pulled from capillaries (Warner Instruments), and their electrode resistance was 0.2 to 1.0 M. After the oocytes had been pricked with microelectrodes filled with 3 M KCl in SOS solution, the bath solution was exchanged with 10 mM Ba²⁺ solution [10 mM Ba(OH)₂, 90 mM NaOH, 1 mM KOH, and 5 mM HEPES, pH 7.4 with methanesulfonic acid]. However, Na⁺ currents were recorded using SOS containing 100 mM NaCl. Barium currents were acquired at 5 kHz and low pass filtered at 1 kHz, and Na⁺ currents were acquired at 20 kHz and low pass filtered at 5 kHz using the pClamp system (Digidata 1320A and pClamp 8; Axon Instruments, Foster City, CA). Data were analyzed with Clampfit software (Axon Instruments) and presented graphically using Prism software (GraphPad Software Inc., San Diego, CA). They are presented as means ± S.E.M. and tested for significance using Student's unpaired t test.

	Results

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Expression and Characterization of Human Ca_v3.2 T-Type Ca²⁺ Channels. Using the two-electrode voltage-clamp method, expression of Ca_v3.2 (_1H) channels in Ca_v3.2 cRNA-injected oocytes was detected as robust transient Ba²⁺ currents (>500 nA) elicited by a test potential of -20 mV from a holding potential of -90 mV (Fig. 1A). In contrast, measurable transient Ba²⁺ currents were not detected in H₂O-injected or uninjected oocytes (data not shown). The Ca_v3.2 currents elicited by a voltage protocol consisting of serial test potentials that were increased by 10 mV from a holding potential of -90 mV displayed the typical biophysical properties of T-type channel currents, such as a low-voltage threshold of approximately -60 mV for activation, fast activation and inactivation, criss-crossing pattern between current traces, peak current at -20 mV, and reversal potential around +40 mV. These properties are identical to those described in previous reports (Lee et al., 1999; Park et al., 2003).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Effect of 10 µM forskolin on human Cav3.2 T-type channel. A, representative current traces before (a) and 30 min after forskolin application (b) are overlapped. B, time course of forskolin-mediated stimulation of Cav3.2 channel activity. A representative time course is plotted. The Cav3.2 current was evoked by a test potential of -20 mV from a holding potential of -90 mV every 20 s, using 10 mM Ba2+ as charge carrier. Current amplitude was normalized to the control current amplitude before forskolin application. Cav3.2 activity was increased by 40 ± 4% in response to 10 µM forskolin within 30 min (n = 20). C, current-voltage relationship. Currents were evoked by depolarizing voltage steps in 10-mV increments from -70 to +40 mV from a holding potential of -90 mV. Peak currents obtained during test potentials were normalized to the maximum observed. D, peak currents were normalized to the maximum observed before the application of forskolin (control, ; forskolin application, ).

Augmentation of Ca_v 3.2 T-Type Ca²⁺ Channel Activity by Forskolin. When currents were evoked by a test potential to -20 mV from a holding potential of -90 mV every 20 s, no significant run-up or run-down was observed over 30 min (data not shown). However, application of 10 µM forskolin, an adenylyl cyclase stimulant, increased the amplitude of the Ca_v3.2 currents with a delay of approximately 2 to 3 min. The Ca_v3.2 current amplitude increased continuously over 30 min (Fig. 1B). Forskolin-induced stimulation of current amplitude reached a maximum level (saturation) within 70 min, where they were stable or ran down slowly (n = 3; data not shown). Representative traces before and 30 min after addition of 10 µM forskolin effect were overlapped for comparison (Fig. 1A). On average, 10 µM forskolin increased the amplitude of Ca_v3.2 currents by 40 ± 4% within 30 min (n = 20). Comparison of current-voltage (I-V) relationships before and 30 min after forskolin addition showed that -fold stimulations at different potentials were similar and that the I-V curve was not shifted (Fig. 1, C and D).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Biophysical properties of human Cav3.2 T-type channels before and after forskolin treatment. A, steady-state activation and inactivation curves before () and 30 min after () application of forskolin. Activation curves were obtained by plotting chord conductances (G), which were calculated by dividing the current amplitude by the driving force. Smooth curves represent the fit of the data to the Boltzman equation (G = 1/[1 + exp(V50 - V)/k]), where V50 is the half-activation voltage, and k is a slope factor. B, time course of recovery from inactivation before and 30 min after forskolin application. Data are averaged and fitted by single exponential association. C and D, activation and inactivation of the current traces were obtained by fitting them with two exponentials simultaneously, one for the activation kinetics and the other for the inactivation kinetics.

Forskolin Stimulates Ca_v3.2 Activity via PKA. We tested whether the forskolin effect occurs via conversion of ATP to cAMP because of stimulation of adenylyl cyclase and subsequent activation of PKA. Indeed, application of 200 µM 8-Br-cAMP, a membrane-permeable cAMP, enhanced the peak amplitude of Ca_v3.2 currents by 39 ± 8% (n = 4) over 30 min. The stimulation profile of 8-Br-cAMP, including delay, extent of stimulation, and time course, was very similar to that of forskolin (Fig. 3). We next tested whether the forskolin effect was mediated by activation of PKA. When oocytes were preincubated in SOS containing 20 µM H89, a PKA-specific inhibitor, superfusion of 10 µM forskolin enhanced the amplitude of Ca_v3.2 currents by only 5% (Fig. 4A). Likewise, the forskolin enhancement effect was almost abolished by preincubation with 200 µM RpBrcAMPs, a competitive antagonist of cAMP binding to PKA (Fig. 4B). Furthermore, the increase in the Ca_v3.2 current amplitude in response to forskolin could be almost completely reversed by subsequent application of 500 nM PKI, a membrane-permeable PKA inhibitor peptide (Fig. 4C). Together, these findings indicate that the stimulation by forskolin arises from activation of the PKA signaling pathway.

View larger version (14K): [in this window] [in a new window]   Fig. 3. 8-Br-cAMP mimics the forskolin effect on human Cav3.2 T-type channels. A, representative time course of the effect of 200 µM 8-Br-cAMP on the peak current amplitude of Cav3.2. Currents were evoked by a test potential of -20 mV from a holding potential of -90 mV every 20 s, and their amplitudes were normalized to the control current amplitude before 8-Br-cAMP treatment. Cav3.2 channel activity increased by 39 ± 8% (n = 4) over 30 min in response to 200 µM 8-Br-cAMP. B, representative current traces before (a) and 30 min after 8-Br-AMP application (b) are overlapped.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Attenuation of the forskolin effect on Cav3.2 channel activity by H89, RpBrcAMPs, and PKI. A, average time courses of the effect of 10 µM forskolin on peak current amplitude of Cav3.2 in the absence (control) and presence of 20 µM H89. Preincubation with 20 µM H89 strongly attenuated forskolin stimulation. The percentage stimulation of Cav3.2 activity by 10 µM forskolin was 40 ± 4% over 30 min (control, ; n = 20). In contrast, preincubation with 20 µM H89 reduced stimulation to 5 ± 3% (; n = 4). B, average time courses of peak current amplitude of Cav3.2. Preincubation with 200 µM RpBrcAMPs abolished the forskolin stimulation effect (n = 4). C, reversal of the forskolin augmentation effect by PKI. Application of 10 µM forskolin enhanced Cav3.2 peak amplitude by 36 ± 4% (n = 4) within 30 min. The forskolin effect was almost reversed by subsequent application of 500 nM PKI. A representative time course of Cav3.2 current amplitude in response to forskolin and washing followed by addition of PKI is plotted.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Stimulation of 5HT7 receptors augments Cav3.2 channel activity via PKA. A, effect of 5-HT stimulation of Cav3.2 channel activity with or without preincubation with 20 µM H89 is plotted against time. Cav3.2 channel activity was recorded in oocytes coinjected with Cav3.2 channel and 5HT7 receptor cRNAs. The voltage protocol was the same as that described in Fig. 1. Stimulation of the 5HT7 receptor with 100 nM 5-HT enhanced Cav3.2 activity by 60 ± 7% over 30 min (; n = 8), and stimulation was significantly reduced to 16 ± 3% by preincubation with 20 µM H89 (; n = 4). B, reversal of the 5-HT stimulation effect by PKI. A representative time course of Cav3.2 current amplitude in response to 100 nM 5-HT and washing followed by the addition of PKI is plotted (n = 8).

In oocytes preincubated with 20 µM H89 followed by 5-HT, Ca_v3.2 current amplitude began to increase without any detectable lag, but the initial increase was not sustained; on average, the percentage augmentation of channel activity was only 16 ± 3% over 30 min (Fig. 5A), showing that 5-HT stimulation effect was strongly inhibited by H89 pretreatment. Consistently, application of 100 nM 5-HT increased Ca_v3.2 peak amplitude by 57 ± 6% (n = 8) within 30 min, and most of the 5-HT augmentation effect could be reversed by subsequent application of 500 nM PKI. A representative time course of Ca_v3.2 peak amplitude in response to 100 nM 5-HT, washing, and 500 nM PKI is shown in Fig. 5B. These findings demonstrate that the 5-HT stimulation effect on Ca_v3.2 current amplitude is mainly due to the PKA pathway.

Localization of the Structural Region(s) Contributing to PKA-Mediated Stimulation. Previous studies have shown that rat Na_v1.4 is not regulated by PKA (Smith and Goldin, 1996, 2000). Accordingly, we first confirmed that rat Na_v1.4 channels expressed in oocytes were not affected by activation of PKA (Fig. 6). We then constructed chimeras of the Ca_v3.2 and Na_v1.4 channels to localize the structural regions(s) required for PKA stimulation. We constructed Ca_v3.2/Na_v1.4_N-term, Ca_v3.2/Na_v1.4_I-II, Ca_v3.2/Na_v1.4_II-III, and Ca_v3.2/Na_v1.4_III-IV by replacing individual cytoplasmic loops of Ca_v3.2 with the corresponding loops of rat Na_v1.4, and Ca_v3.2_C was generated by truncating its carboxyl tail (Fig. 6). The expression levels of the chimeric channels were not significantly different from that of the wild-type Ca_v3.2.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Comparison of forskolin effects on Cav3.2, Nav1.4, and their mutant channels. Schematic diagrams of Cav3.2, its deletion mutant, the loop chimeras, and Nav1.4 are shown on the left. Cav3.2 is illustrated by white cylinders and thin lines, and Nav1.4 by gray cylinders and thick lines. Accordingly, the thick lines of the chimeric channels are derived from Nav1.4. Changes of peak current amplitudes caused by 10 µM forskolin were evaluated within 30 min, and average stimulation percentages are plotted as bar graphs on the right (mean ± S.E.M.; n = 4-6). Significant differences were evaluated using the t test, and differences with P 0.001 are designated by ***. The numbers of oocytes tested are given in parentheses.

The activities of Ca_v3.2/Na_v1.4_N-term, Ca_v3.2/Na_v1.4_I-II, Ca_v3.2/Na_v1.4_III-IV, and Ca_v3.2_C were stimulated by forskolin by 36 ± 5, 34 ± 2, 35 ± 6, and 46 ± 8%, respectively, within 30 min (n = 4-5). The stimulation profiles of the loop chimeras and the C-terminal truncation mutant were similar to that of wild-type Ca_v3.2. In contrast, Ca_v3.2/Na_v1.4_II-III activity was little changed by application of forskolin (n = 6; Fig. 6). On average, it was stimulated by only 2 ± 4% over 30 min, much less than that observed for the wild type (P < 0.001, Student's t test). Together, these results strongly suggest that the II-III loop contains structural element(s) critical for the PKA stimulation.

	Discussion

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Electrophysiological recordings have shown that low threshold T-type currents are mainly present in adrenal glomerulosa cells (Matsunaga et al., 1987; Cohen et al., 1988; Rossier et al., 1993). In situ hybridization analysis has shown that of the three T-type channel isoforms, Ca_v3.2 is the major channel in these cells (Schrier et al., 2001). Lenglet et al. (2002) recently reported that T-type currents in rat glomerulosa were stimulated by activation of 5-HT₇ receptors via the PKA signaling pathway. These findings prompted us to test whether the channel activity of recombinant Ca_v3.2 reconstituted in the Xenopus oocyte system was regulated by PKA, and we found that channel activity was indeed enhanced by forskolin via activation of endogenous PKA.

Most previous workers have reported that T-type Ca²⁺ currents are little affected by PKA. For example, application of isoproterenol, a -adrenergic agonist, had no effect on T-type currents recorded from either rabbit sinoatrial node (Hagiwara et al., 1988), canine atrial myocytes (Bean, 1985), guinea pig ventricular myocytes (Tytgat et al., 1988), rabbit ear artery (Benham and Tsien, 1988), canine Purkinje neuron (Hirano et al., 1989; Tseng and Boyden, 1989), or guinea pig hippocampal CA3 neurons (Fisher and Johnston, 1990). In contrast, T-type Ca²⁺ currents recorded from frog atrial myocytes and rat glomerulosa cells were shown to be increased by the cAMP-PKA pathway (Alvarez and Vassort, 1992; Lenglet et al., 2002). The glomerulosa T-type Ca²⁺ current was increased by 5-HT via activation of the PKA signaling pathway. In contrast, Alvarez and Vassort (1992) reported that T-type Ca²⁺ current in frog heart was increased by cAMP treatment and the initial cAMP-mediated increment could be further enhanced by subsequent application of isoproterenol. The cAMP-mediated response was shown to be relatively slow, whereas the subsequent isoproterenol response was fast. They also displayed that the T-type Ca²⁺ current in response to isoproterenol was biphasically enhanced with fast and slow time courses. The cAMP-PKA pathway seemed to be involved in the slow response, whereas the mechanism (possibly G protein-mediated mechanism) responsible for the fast response remained to be uncovered. The fast and slow increment pattern of Ca_v3.2 channel activity by 5-HT shown in this study was somewhat similar to the isoproterenol regulation pattern of frog T-type Ca²⁺ current. It is also similar to the case of the isoproterenol regulation of frog T-type channel current that the up-regulation of Ca_v3.2 current activity in response to forskolin or 5-HT was obtained from oocytes of 11 of 15 frogs. On the contrary, Ca_v3.2 T-type channel currents of oocytes isolated from the other four frogs did not show any enhancement to those drugs. Together, these different regulation results suggest that the PKA regulation effects on T-type channel currents can be variable between tissues expressing T-type channels.

Native L-type Ca²⁺ currents recorded in cardiac myocytes were strongly up-regulated by PKA, whereas recombinant cardiac L-type Ca²⁺ channel currents recorded in HEK293 cells were essentially unaffected (Zong et al., 1995; Mikala et al., 1998). This discrepancy was resolved by the finding that stimulation of cardiac L-type channel currents depended on the presence or absence of a PKA-anchoring protein (AKAP), which played a crucial role in localizing PKA near to the plasma membrane (Gao et al., 1997; Fraser et al., 1998). However, AKAP is not likely to be a critical factor affecting the regulation of T-type channel activity, because T-type currents are insensitive to PKA in cardiac myocytes, in which AKAP is expressed (Gray et al., 1998). The different effects on T-type channels may depend on the presence or absence of unidentified proteins that prevent PKA from interacting with the channels. A related possibility is that T-type currents are up-regulated by activation of PKA via phosphorylation of an accessory subunit rather than the Ca_v3.2 ₁ subunit itself, as shown for K_v1.5 whose regulation by PKA is reported to be via phosphorylation of K_v1.3 (Kwak et al., 1999). Therefore, further efforts should be made to identify regulatory proteins or auxiliary subunits of the T-type channel ₁ subunit.

We localized a structural region contributing to the augmentation of Ca_v3.2 activity to the II-III loop of the Ca_v3.2. To identify a specific locus for phosphorylation by PKA in the II-III loop, we tested the role of Thr1055, Ser1133, and Ser1134 by making point mutation converting them individually into Ala because these sites are found in the motifs [Arg-Arg-X-Ser/Thr or Arg-X-(X)-Ser/Thr] known to be phosphorylated by PKA and are conserved in the three T-type channel isoforms. All of the mutant channels were regulated by forskolin, and their regulatory profiles were similar to that of wild-type Ca_v3.2, suggesting that these sites do not contribute to the PKA-mediated stimulation. In addition to the three sites examined, there are eight more putative sites fitting the consensus motifs for PKA phosphorylation. Their involvement remains to be investigated by making individual point mutations.

In summary, we have shown that Ca_v3.2 channel activity can be up-regulated by PKA, and we were able to reconstitute the PKA signaling pathway augmenting T-type channel activity in Xenopus oocytes. In addition, we localized the structural region involved in the PKA stimulation to the II-III loop. The PKA stimulation of cloned T-types Ca²⁺ channels in the Xenopus oocyte system demonstrated here may contribute to understanding T-type channel regulation by neurotransmitters and hormones.

	Footnotes

 
This work was supported by the Korea Research Foundation Grants KRF-2005-015-C00403 and KRF-2005-042-C00058 funded by the Korean government.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.101402.

ABBREVIATIONS: PKA, cAMP-dependent protein kinase A; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; 5-HT, 5-hydroxytryptamine; 8-Br-cAMP, 8-bromo-cAMP; RpBrcAMPs, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer; PKI, protein kinase A inhibitor peptide; PCR, polymerase chain reaction; I-V, current-voltage; AKAP, protein kinase A-anchoring protein.

¹ These authors contributed equally to this work.

Address correspondence to: Dr. Jung-Ha Lee, Department of Life Science, Sogang University, Mapo-Gu, Sinsu-Dong 1, Seoul 121-742, Korea. E-mail: jhleem{at}sogang.ac.kr

	References

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