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• W2009673797 abstract "Modulation of low voltage-activated CaV3 T-type calcium channels remains poorly characterized compared with high voltage-activated CaV1 and CaV2 calcium channels. Notably, it is yet unresolved whether CaV3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in CaV3.1, CaV3.2, and CaV3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature (∼30–37 °C) but not at room temperature (∼22–27 °C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated CaV2.3 currents. We also report that neither CaV3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of CaV3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of CaV3 T-channels and further highlight the key role of the physiological temperature in the effects described. Modulation of low voltage-activated CaV3 T-type calcium channels remains poorly characterized compared with high voltage-activated CaV1 and CaV2 calcium channels. Notably, it is yet unresolved whether CaV3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in CaV3.1, CaV3.2, and CaV3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature (∼30–37 °C) but not at room temperature (∼22–27 °C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated CaV2.3 currents. We also report that neither CaV3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of CaV3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of CaV3 T-channels and further highlight the key role of the physiological temperature in the effects described. Voltage-gated Ca2+ channels (VGCCs) 5The abbreviations used are: VGCCs, voltage-gated Ca2+ channels; PKC, protein kinase C; PKA, protein kinase A; PKG, protein kinase G; CHO, Chinese hamster ovary; GFP, green fluorescent protein; HA, hemagglutinin; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; Bt2cAMP, dibutyryl cAMP; Bt2cGMP, dibutyryl cGMP. are unique among voltage-gated ion channels because the permeant Ca2+ ion also acts as an intracellular second messenger, triggering diverse cellular functions (1Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1766) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4491) Google Scholar). VGCCs are therefore implicated in neuronal and cardiac excitability as well as in muscle contraction, neurotransmitter release or hormone secretion, and gene expression (1Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1766) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4491) Google Scholar, 3Noma A. Jpn. Heart J. 1996; 37: 673-682Crossref PubMed Scopus (46) Google Scholar, 4Striessnig J. Cell. Physiol. Biochem. 1999; 9: 242-269Crossref PubMed Scopus (164) Google Scholar, 5Kamp T.J. Hell J.W. Circ. Res. 2000; 87: 1095-1102Crossref PubMed Scopus (502) Google Scholar, 6Tedford H.W. Zamponi G.W. Pharmacol. Rev. 2006; 58: 837-862Crossref PubMed Scopus (193) Google Scholar). Thus, the modulation of VGCCs plays a pivotal role in the control of cardiac and brain activities. VGCCs are divided into three families: the L-type channels (CaV1 family); the neuronal N-, P/Q-, and R-type channels (CaV2 family); and the T-type channels (CaV3 family) (7Ertel E.A. Campbell K.P. Harpold M.M. Hofmann F. Mori Y. PerezReyes E. Schwartz A. Snutch T.P. Tanabe T. Birnbaumer L. Tsien R.W. Catterall W.A. Neuron. 2000; 25: 533-535Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar). Although the molecular mechanisms implicated in the modulation of high voltage-activated Ca2+ channels of the CaV1 and CaV2 families are beginning to be unraveled (mainly, protein kinases for CaV1 channels and βγ-subunits of G proteins and protein kinase C (PKC) for CaV2 channels) (6Tedford H.W. Zamponi G.W. Pharmacol. Rev. 2006; 58: 837-862Crossref PubMed Scopus (193) Google Scholar, 8Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1957) Google Scholar), those implicated in low voltage-activated CaV3 T-type channel regulation remain debated (9Chemin J. Traboulsie A. Lory P. Cell Calcium. 2006; 40: 121-134Crossref PubMed Scopus (71) Google Scholar). Some transduction pathways mediate either decreases or increases in native T-currents, depending on the tissues/species studied and/or on the recording conditions (9Chemin J. Traboulsie A. Lory P. Cell Calcium. 2006; 40: 121-134Crossref PubMed Scopus (71) Google Scholar). This apparent complexity could be explained by the existence of three T-channels (CaV3.1 or α1G, CaV3.2 or α1H, and CaV3.3 or α1I), which include different splice variant isoforms with specific tissue patterns and developmental expression (10Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1350) Google Scholar). Additional regulatory subunits of T-channels might also be involved in their modulation, but the native composition of T-channels remains unknown (10Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1350) Google Scholar). Therefore, molecular studies on recombinant T-channels are required to clarify their modulation. Recent studies on recombinant T-channels have highlighted the complexity of their regulation (9Chemin J. Traboulsie A. Lory P. Cell Calcium. 2006; 40: 121-134Crossref PubMed Scopus (71) Google Scholar). On one hand, as for CaV2 channels, CaV3.2 currents are inhibited by βγ-subunits of G proteins in mammalian cells (11Wolfe J.T. Wang H. Howard J. Garrison J.C. Barrett P.Q. Nature. 2003; 424: 209-213Crossref PubMed Scopus (125) Google Scholar, 12DePuy S.D. Yao J. Hu C. McIntire W. Bidaud I. Lory P. Rastinejad F. Gonzalez C. Garrison J.C. Barrett P.Q. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14590-14595Crossref PubMed Scopus (38) Google Scholar). In contrast with CaV2 currents, this mechanism cannot be generalized because this modulation is restricted to CaV3.2 and specifically occurs with a Gβγ dimer containing β2-subunits (11Wolfe J.T. Wang H. Howard J. Garrison J.C. Barrett P.Q. Nature. 2003; 424: 209-213Crossref PubMed Scopus (125) Google Scholar, 12DePuy S.D. Yao J. Hu C. McIntire W. Bidaud I. Lory P. Rastinejad F. Gonzalez C. Garrison J.C. Barrett P.Q. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 14590-14595Crossref PubMed Scopus (38) Google Scholar). On the other hand, as observed with CaV1 channels, CaV3 currents are increased by protein kinase activation (13Wolfe J.T. Wang H. Perez-Reyes E. Barrett P.Q. J. Physiol. (Lond.). 2002; 538: 343-355Crossref Scopus (71) Google Scholar, 14Welsby P.J. Wang H. Wolfe J.T. Colbran R.J. Johnson M.L. Barrett P.Q. J. Neurosci. 2003; 23: 10116-10121Crossref PubMed Google Scholar, 15Yao J. Davies L.A. Howard J.D. Adney S.K. Welsby P.J. Howell N. Carey R.M. Colbran R.J. Barrett P.Q. J. Clin. Investig. 2006; 116: 2403-2412PubMed Google Scholar, 16Park J.Y. Jeong S.W. Perez-Reyes E. Lee J.H. FEBS Lett. 2003; 547: 37-42Crossref PubMed Scopus (33) Google Scholar, 17Kim J.A. Park J.Y. Kang H.W. Huh S.U. Jeong S.W. Lee J.H. J. Pharmacol. Exp. Ther. 2006; 318: 230-237Crossref PubMed Scopus (50) Google Scholar, 18Park J.Y. Kang H.W. Moon H.J. Huh S.U. Jeong S.W. Soldatov N.M. Lee J.H. J. Physiol. (Lond.). 2006; 577: 513-523Crossref Scopus (57) Google Scholar). Yet again, kinase effects on T-currents appear complex (9Chemin J. Traboulsie A. Lory P. Cell Calcium. 2006; 40: 121-134Crossref PubMed Scopus (71) Google Scholar) because protein kinase A (PKA)- and PKC-mediated T-current increase has been observed in Xenopus oocytes but not in mammalian cells expressing CaV3 currents (11Wolfe J.T. Wang H. Howard J. Garrison J.C. Barrett P.Q. Nature. 2003; 424: 209-213Crossref PubMed Scopus (125) Google Scholar, 16Park J.Y. Jeong S.W. Perez-Reyes E. Lee J.H. FEBS Lett. 2003; 547: 37-42Crossref PubMed Scopus (33) Google Scholar, 17Kim J.A. Park J.Y. Kang H.W. Huh S.U. Jeong S.W. Lee J.H. J. Pharmacol. Exp. Ther. 2006; 318: 230-237Crossref PubMed Scopus (50) Google Scholar, 18Park J.Y. Kang H.W. Moon H.J. Huh S.U. Jeong S.W. Soldatov N.M. Lee J.H. J. Physiol. (Lond.). 2006; 577: 513-523Crossref Scopus (57) Google Scholar, 19Zhang Y. Cribbs L.L. Satin J. Am. J. Physiol. 2000; 278: H184-H193PubMed Google Scholar). In this context, it is interesting to note that protein kinase effects on native VGCCs were first described in heart frog cells and appear more robust in these cells compared with those observed in mammalian tissues (20Vassort G. Rougier O. Garnier D. Sauviat M.P. Coraboeuf E. Gargouil Y.M. Pfluegers Arch. 1969; 309: 70-81Crossref PubMed Scopus (163) Google Scholar, 21Bean B.P. Nowycky M.C. Tsien R.W. Nature. 1984; 307: 371-375Crossref PubMed Scopus (239) Google Scholar, 22Tsien R.W. Bean B.P. Hess P. Lansman J.B. Nilius B. Nowycky M.C. J. Mol. Cell. Cardiol. 1986; 18: 691-710Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 23Alvarez J.L. Vassort G. J. Gen. Physiol. 1992; 100: 519-545Crossref PubMed Scopus (54) Google Scholar). Thus, we reasoned that these differences in kinase modulation could involve the temperature at which the experiments were performed, i.e. mostly at room temperature. Although room temperature is within the physiological range for amphibian cells, it is far below that required by mammalian cells. In this study, we provide new data on PKA and PKC activation in the function of temperature that clearly establish the role of these two kinases, but not of protein kinase G (PKG), in the modulation of CaV3 T-currents in various mammalian cell lines. Cell Culture and Transfection Protocols—tsA-201 cells and a Chinese hamster ovary (CHO) cell line stably expressing CaV3.2 channels (CHO-CaV3.2; a generous gift from Dr. Emmanuel Bourinet) were cultivated in Dulbecco's modified Eagle's medium and Ham's F-12 medium, respectively, supplemented with GlutaMAX and 10% fetal bovine serum (Invitrogen). Neomycin (0.6 mg/ml; Invitrogen) was added to the CHO-CaV3.2 cell medium. tsA-201 cell transfection was performed using jetPEI (Qbiogen, Inc.) according to the manufacturer's protocol (4 μl of jetPEI for ∼1.5 μg of DNA/35-mm Petri dish) with a DNA mixture containing 0.5% of a green fluorescent protein (GFP) plasmid and 99.5% of one of the pcDNA3 plasmid constructs that code for the human CaV3.1a, CaV3.2, and CaV3.3 T-channel isoforms (24Chemin J. Nargeot J. Lory P. J. Biol. Chem. 2007; 282: 2314-2323Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). CaV2.1-hemagglutinin (HA) (25Mullner C. Broos L.A. van den Maagdenberg A.M. Striessnig J. J. Biol. Chem. 2004; 279: 51844-51850Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and CaV2.3 (rat brain E-II) (26Stea A. Soong T.W. Snutch T.P. Neuron. 1995; 15: 929-940Abstract Full Text PDF PubMed Scopus (194) Google Scholar) were transfected under the same conditions with a mixture containing the α2δ1- and β4-subunits at a 2:1:1 ratio. Two days after, cells were then dissociated with Versen (Invitrogen) and plated at ∼35 × 103 cells/35-mm Petri dish. Electrophysiological recordings were performed the following day. Electrophysiological Recordings—Macroscopic currents were recorded at room temperature (∼22 °C) by the whole-cell patch clamp technique using an Axopatch 200B amplifier (Axon Instruments). The extracellular solution contained 135 mm NaCl, 20 mm tetraethylammonium chloride, 2 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH adjusted to 7.4 with KOH, ∼330 mosm). Borosilicate glass pipettes have a typical resistance of 1.5–2.5 megaohms when filled with an internal solution containing 140 mm CsCl, 10 mm EGTA, 10 mm HEPES, 3 mm MgATP, 0.6 mm NaGTP, and 2 mm CaCl2 (pH adjusted to 7.2 with KOH, ∼315 mosm). Recordings were filtered at 2 kHz. Data were analyzed using pCLAMP9 (Axon Instruments) and GraphPad Prism software. Student' t test or one-way analysis of variance combined with a Newman-Keuls post-test were used to compare the different values, which were considered significant at p < 0.05. Results are presented as the means ± S.E., and n is the number of cells used. Imaging of PKC Translocation—To study PKC translocation as a function of temperature, we generated a human PKCβ1 construct with the C terminus fused to GFP using the pEGFP-N1 plasmid (Clontech). PKCβ1-GFP was inserted into pcDNA4 (Invitrogen) and transfected into CHO-CaV3.2 cells. Two days after, cells were plated onto 12-mm glass coverslips. The following day, phorbol 12-myristate 13-acetate (PMA) treatments were performed (100 nm in the culture medium for 10 min at 37 °C or at room temperature), and cells were then fixed for 20 min in 4% paraformaldehyde. Digital images were acquired on a Leica microscope and further analyzed using Adobe Photoshop. Luminometric Analysis of HA-tagged CaV3.2 Channels—tsA-201 cells were cultured in 24-well plates and transfected with a GFP-CaV3.2-HA construct (27Dubel S.J. Altier C. Chaumont S. Lory P. Bourinet E. Nargeot J. J. Biol. Chem. 2004; 279: 29263-29269Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Two days after transfection, PMA treatments were performed (100 nm in the culture medium for 30 or 60 min at 37 °C), and cells were then rinsed with phosphate-buffered saline (PBS) and fixed for 5 min in 4% paraformaldehyde. After three PBS washes, half of the wells were permeabilized with 0.1% Triton X-100 for 5 min and rinsed three times with PBS. Cells were then incubated for 30 min in blocking solution (PBS plus 1% fetal bovine serum). The GFP-CaV3.2-HA protein was detected using a rat anti-HA monoclonal antibody (1:1000 dilution; clone 3F10, Roche Diagnostics) after incubation for 1 h at room temperature. After four washes with PBS plus 1% fetal bovine serum for 10 min, cells were incubated for 30 min with horseradish peroxidase-conjugated goat anti-rat secondary antibody (1:1000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were rinsed four times with PBS for 10 min before addition of SuperSignal enzyme-linked immunosorbent assay Femto maximum sensitivity substrate (Pierce). Luminescence was measured using a VICTOR2 luminometer (PerkinElmer Life Sciences), and the protein amount in each well was then measured using the BCA assay (Pierce) to normalize the measurements. All data were normalized to the level of signal obtained for incubation with the control medium (1:10,000 Me2SO). Four independent sets of transfection experiments were performed under each condition, and the results are presented as the means ± S.E. In Vitro PKA Assays—tsA-201 cells transfected with HA-tagged CaV3.2 or CaV2.1 were lysed in Nonidet P-40 buffer (1% Nonidet P-40, 150 mm NaCl, and 10 mm Tris-HCl (pH 7.6) supplemented with a protease inhibitor mixture (Roche Diagnostics)) for 30 min on ice. Lysates were centrifuged at 10,000 × g for 20 min, precleared for 1 h with fetal calf serum-Sepharose beads, and incubated overnight with specific antibodies and protein A-Sepharose beads (Amersham Biosciences). Beads were washed three times with Nonidet P-40 buffer before PKA assays. In vitro PKA assays were performed on Sepharose beads for 30 min at 30 °C with 10 units of the PKA catalytic subunit (Sigma) in 40 μl of medium containing 50 mm Tris-HCl, 10 mm MgCl2, 10 mm HEPES, 10 mm dithiothreitol, 1 mm Na3VO4, 1 mm MgATP, 0.15 mm CaCl2, 500 μm okadaic acid, and 10 μCi of [γ-32P]ATP (Amersham Biosciences). After three washes with cold Nonidet P-40 buffer, standard SDS-PAGE was performed, and phosphorylated proteins were analyzed using a PhosphorImager. Chemical Reagents—All compounds (which were from Sigma, except Gö 6976, which was from BIOMOL International LP) were dissolved in water (1 mm), except PMA, 4α-PMA, and Gö 6976, which were dissolved in Me2SO (1 mm); aliquoted; and kept at –80 °C in the dark. Control experiments were carried out using the solvent alone. To investigate the effects of protein kinase activation in mammalian cells expressing low voltage-activated T-currents, various protein kinase activators (or the control solution) were incubated with cells at 37 °C or at room temperature (∼22 °C) for 10 min before electrophysiological recordings, which were performed at room temperature in either the presence or absence of the activator (Fig. 1, A and B, insets). Under these conditions, the PKC activator PMA (100 nm) induced a strong increase in the CaV3.1 currents (∼100%, p < 0.001, n > 70) when preincubated at 37 °C (Fig. 1A). Similar results were obtained for CaV3.2 and CaV3.3 channels because PMA induced an ∼105% increase (p < 0.001, n > 90) in CaV3.2 currents and had a maximal effect on CaV3.3 currents (∼145%, p < 0.001, n > 100) (Fig. 1C). Interestingly, we found that PMA had no significant effect when incubated at room temperature (up to 1 h of incubation; p > 0.05 and n > 30 for each CaV3 current) (Fig. 1, B–D). We then evaluated the PMA effects on CaV3.3 currents during incubation at several intermediate temperatures (27, 30, and 32 °C). We found that the PMA effects gradually developed at 30 and 32 °C (∼60 and 100% increases, respectively; p < 0.05; n > 40) (Fig. 2), whereas no effect was observed at 27 °C (p > 0.05, n > 34) (Fig. 2). The PMA effects as a function of temperature can be described by a sigmoidal Hill equation (Fig. 2), which indicates that the temperature producing PMA half-effects is ∼30.5 °C and the Hill slope ∼18.9, indicating a strong temperature dependence of PMA effects. We also investigated the temperature dependence of the PMA effects on high voltage-activated CaV2.3 currents, which are increased by PMA at room temperature in Xenopus oocytes (26Stea A. Soong T.W. Snutch T.P. Neuron. 1995; 15: 929-940Abstract Full Text PDF PubMed Scopus (194) Google Scholar). As observed for CaV3 currents, we found that 100 nm PMA induced a strong increase in CaV2.3 currents in human embryonic kidney 293 cells when incubated at 37 °C (∼150%, p < 0.001, n > 30) (Fig. 3, A and B), whereas PMA has no effect when incubated at room temperature (p > 0.05, n > 35) (Fig. 3C).FIGURE 2Temperature dependence of PMA effects on CaV3.3 T-currents in transiently transfected tsA-201 cells. Shown is the effect of 100 nm PMA or the control solution (10-min incubation) on CaV3.3 currents at several temperatures, i.e. at 22 (room temperature), 27, 30, 32, and 37 °C. Electrophysiological experiments were performed at room temperature during the following 50 min in the presence of PMA or the control solution. Currents were elicited by a –45-mV depolarization of 450-ms duration applied immediately after the whole-cell configuration from a holding potential of –80 mV. Increases in CaV3.3 currents as a function of temperature were fitted with a sigmoidal Hill equation, where T-current increase (Y) = Ymin + (Ymax–Ymin)/(1 + (T0.5/T)Hill slope), where T is temperature. The fit gave a T0.5 value of 30.54 °C and a Hill slope of 18.87.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3PMA induces an increase in CaV2.3 currents in transiently transfected tsA-201 cells at 37 °C but not at room temperature (∼22 °C). A–C, effect of 100 nm PMA or the control solution (Ctrl) on CaV2.3 currents when incubated for 10 min at 37 °C (A and B) or at room temperature (R.T.; C) before electrophysiological experiments, which were performed at room temperature during the following 50 min in the presence of PMA or the control solution. Currents were elicited by a +10-mV depolarization of 500-ms duration applied immediately after the whole-cell configuration from a holding potential of –80 mV. ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been described that PKC activation is associated with a redistribution of its subcellular localization (28Dorn G.W. II Mochly-Rosen Annu. Rev. Physiol. 2002; 64: 407-429Crossref PubMed Scopus (125) Google Scholar). We asked whether this phenomenon is altered in mammalian cells at a non-physiological temperature (Fig. 4). To this end, we generated a GFP-tagged PKCβ1 construct (PKCβ1-GFP; see “Materials and Methods”) to visualize PMA-induced PKC translocation in transfected mammalian cells (Fig. 4, B and C). As described for PKC of the classical family (28Dorn G.W. II Mochly-Rosen Annu. Rev. Physiol. 2002; 64: 407-429Crossref PubMed Scopus (125) Google Scholar), we found that 100 nm PMA induced PKC translocation to the plasma membrane in mammalian CHO cells when incubated for 10 min at 37 °C (Fig. 4B). However, PMA had no effect on PKC translocation when incubated at room temperature (Fig. 4D). We next investigated whether PMA effects on CaV3 currents are specific to PKC activation (Fig. 5). To assess the involvement of PKC, we used 4α-PMA (100 nm), a PMA analog inactive on PKC, and chelerythrine or Gö 6976 (both at 1 μm), two selective inhibitors of PKC (Fig. 5). We found that 4α-PMA had no significant effect on the three CaV3 currents (p > 0.05 and n > 25 for each CaV3 current), whereas both chelerythrine and Gö 6976 suppressed PMA effects (p > 0.05 and n > 25 for each CaV3 current). In addition, we found no basal effect of chelerythrine on the three CaV3 currents (p > 0.05 and n > 60 for each CaV3 current) (Fig. 5, A–C). To estimate whether PKC activation-induced increase in T-currents is associated with changes in macroscopic biophysical properties, we next performed steady-state activation and inactivation protocols (Fig. 6, A and B, insets). As shown for CaV3.1 (Fig. 6, A–D), PMA induced no significant change in steady-state activation and inactivation properties. For CaV3.1, current-voltage curves (Fig. 6C) indicated steady-state activation V0.5 values of –48.6 ± 1.4 mV (n = 10) under control conditions and –47.6 ± 1.3 mV (n = 13, p > 0.05) after PMA treatment, whereas slope values were 4.1 ± 0.4 mV (n = 10) under control conditions and 4.3 ± 0.4 mV (n = 13, p > 0.05) after PMA treatment. Similarly, steady-state inactivation properties were not significantly modified by PKC activation (Fig. 6D) because V0.5 values were –70.7 ± 0.7 mV (n = 10) under control conditions and –71.8 ± 0.6 mV (n = 11, p > 0.05) after PMA treatment, whereas slope values were 4.7 ± 0.2 mV (n = 10) under control conditions and 4.1 ± 0.2 mV (n = 11, p > 0.05) after PMA treatment. In the same way, neither the steady-state activation nor inactivation properties of the CaV3.2 (n > 10 under each condition, p > 0.05) (Fig. 6E) and CaV3.3 (n > 10 under each condition, p > 0.05) (Fig. 6F) currents were significantly affected by PMA treatment. We also investigated whether the surface expression of CaV3 channels in mammalian tsA-201 cells is modulated by PKC activation (supplemental Fig. 1). To this end, we used a CaV3.2 channel construct containing an extracellular HA tag, which allowed its surface (non-permeabilized condition) and total expression (permeabilized condition) (supplemental Fig. 1A) to be measured by enzyme-linked immunosorbent assay/luminometry. We found that PMA treatment did not induce significant changes in both membrane expression (supplemental Fig. 1B) and total expression (supplemental Fig. 1C) of CaV3.2-HA channels after 30 min or 1 h of treatment at 37 °C. Also, protein kinase activation did not influence the ratio of membrane expression to total expression, which was ∼20% under all conditions (supplemental Fig. 1D), further indicating that surface expression of CaV3.2-HA channels is not altered. We next explored whether, as observed for PKC, cyclic nucleotide-dependent protein kinases modulate T-currents in mammalian cells at physiological temperatures. To investigate the effects of PKA, we used dibutyryl cAMP (Bt2cAMP), a membrane-permeant analog of cAMP (Fig. 7). We found that Bt2cAMP induced an increase in the three CaV3 T-currents when incubated at 37 °C (Fig. 7, A–C) but had no significant effect when incubated at room temperature (p > 0.05 and n > 30 for each CaV3 current) (Fig. 7, B–D). As observed above with PMA, Bt2cAMP produced a stronger effect on CaV3.3 currents (∼140% increase, p < 0.001, n > 60) compared with CaV3.2 currents (∼70% increase, p < 0.001, n > 80) and CaV3.1 currents (∼55% increase, p < 0.001, n > 80) (Fig. 7C). Furthermore, we observed a similar temperature dependence using monobutyryl cAMP (the bioactive product of Bt2cAMP acting on PKA). Indeed, monobutyryl cAMP treatment increased CaV3.3 currents at 37 °C (∼110% increase, p < 0.01, n > 35) but not at room temperature (p > 0.05, n > 40). These latter results demonstrate that the temperature dependence described here does not involve endogenous esterases or amidases, which convert Bt2cAMP into monobutyryl cAMP (for review, see Ref. 29Schwede F. Maronde E. Genieser H. Jastorff B. Pharmacol. Ther. 2000; 87: 199-226Crossref PubMed Scopus (214) Google Scholar). We next probed the involvement of PKA activation in the Bt2cAMP effects using the specific PKA inhibitor KT5720. KT5720 suppressed Bt2cAMP-induced increases in T-currents (p > 0.05 and n > 20 for each CaV3 current) (Fig. 8, A–C) but had no effect on basal T-currents (p > 0.05 and n > 20 for each CaV3 current). It should be noted that in previous experiments, we used the PKA inhibitor H-89, but we observed that although this compound abolished Bt2cAMP effects, it had strong direct inhibitory effects at micromolar concentrations on the three CaV3 currents and especially on CaV3.2 (data not shown). We further investigated whether PKA effects would involve a direct phosphorylation of CaV3 channels. To this end, we took advantage of a commercially available PKA catalytic subunit that is constitutively active and that allows in vitro kinase assays of immunoprecipitated HA-tagged CaV3.2 channels (Fig. 8D). We found that PKA induced phosphorylation of CaV3.2 channels, but not of CaV2.1 channels, which are insensitive to PKA (6Tedford H.W. Zamponi G.W. Pharmacol. Rev. 2006; 58: 837-862Crossref PubMed Scopus (193) Google Scholar, 8Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1957) Google Scholar) and were used here as a negative control (Fig. 8D).FIGURE 8Involvement of PKA in Bt2cAMP-induced increases in the three CaV3 currents and direct phosphorylation of CaV3.2 channels by PKA. A–C, summary of the data obtained for CaV3.1, CaV3.2, and CaV3.3, respectively, after 10 min of incubation at 37 °C of Bt2cAMP (dB-cAMP; 1 mm) and/or KT5720 (a PKA inhibitor; 0.5 μm). KT5720 was applied at least 4 h at 1 μm prior to the 37 °C incubation protocols. Currents were elicited by a –45-mV depolarization (200-ms duration for CaV3.1 and CaV3.2 currents and 450-ms duration for CaV3.3 currents) applied immediately after the whole-cell configuration from a holding potential of –80 mV. Ctrl, control. D, effects of the constitutively active PKA catalytic subunit on immunoprecipitated HA-tagged CaV2.1 and CaV3.2 channels in in vitro phosphorylation assays. In vitro phosphorylation assays were performed at 30 °C using [γ-32P]ATP, and 32P-labeled proteins were quantified using a PhosphorImager after standard SDS-PAGE. The presence of HA-tagged CaV2.1 and CaV3.2 channels was further confirmed by Western blotting (not shown). ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast with the results obtained for PKA activation, the activation at physiological temperature of PKG with dibutyryl cGMP (Bt2cGMP) had no significant effect on the three CaV3 currents (p > 0.05 and n > 40 for each CaV3 current) (supplemental Fig. 2, A and B). Furthermore, we treated cells with the specific PKG inhibitor KT5823 (supplemental Fig. 2B) and found that this compound had no significant effect on the three CaV3 current densities (p > 0.05 and n > 20 for each CaV3 current) (supplemental Fig. 2B). Finally, we further investigated the effect of PKC, PKA, and PKG activation in a CHO cell line stably expressing CaV3.2 currents (Fig. 9). In these cells, we found that PMA and Bt2cAMP (but not Bt2cGMP) induced an increase in CaV3.2 currents when incubated at physiological temperature (Fig. 9A). As observed above in transiently transfected tsA-201 cells, PMA induced stronger effects on CaV3.2 currents (∼125% increase) compared with Bt2cAMP (∼50% increase, p < 0.001, and n > 30 under each condition) (Fig. 9B). However, PMA and Bt2cAMP had no significant effect when incubated at room temperature (up to 1 h of incubation; p > 0.05 and n > 24 under each condition) (Fig. 9C). The main findings of our study are that recombi" @default.
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• W2009673797 title "Temperature-dependent Modulation of CaV3 T-type Calcium Channels by Protein Kinases C and A in Mammalian Cells" @default.
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