Trafficking of L-type Calcium Channels Mediated by the Postsynaptic Scaffolding Protein AKAP79

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and β2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events. Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and β2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events. protein kinase A A-kinase anchoring protein polyprolines open probability wild-type hemagglutinin tetraethylammonium α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Voltage-gated calcium channels (VGCCs) are transmembrane proteins involved in the regulation of cellular excitability and Ca2+ homeostasis in excitable and non-excitable cells (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar). They play a key role in numerous cellular functions including enzyme activation, muscle contraction, neurotransmitter release, and gene transcription. Molecular cloning has led to the isolation and functional expression of a number of subunits that form Ca2+ channels. Based on primary structure homology, these subunits are separated into three different families (2Ertel E.A. Campbell K.P. Harpold M.M. Hofmann F. Mori Y. Perez-Reyes 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). High voltage-activated channels are comprised of the L-types (CaV1) and the N-, P/Q-, and R-types (CaV2). The third family (CaV3) is comprised of the members of the low voltage-activated/T-type Ca2+ channels. Although primarily gated by fluctuations of membrane potential, an essential aspect of the function of these channels is their capacity to respond to extracellular signals via membrane receptors and intracellular second messengers that, in turn, alter channel activity. As for many ionic channels, growing evidence indicates that the specificity and speed of these regulations require a promiscuous organization of the constitutive channel subunits with membrane receptors and complexes of intracellular molecules. In that context, the role of scaffolding proteins in orchestrating these networks is crucial. Accordingly, the regulation of voltage-gated calcium channels by anchored pools of protein kinases is a key factor in controlling intracellular calcium levels. Efficient phosphorylation is accomplished through formation of kinase-channel complexes. For example, protein kinase A (PKA)1 is anchored near L-type calcium channels by different AKAPs in brain, skeletal muscle, smooth muscle, and myocardium (3Johnson B.D. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11492-11496Crossref PubMed Scopus (157) Google Scholar, 4Zhong J. Hume J.R. Keef K.D. Am. J. Physiol. 1999; 277 (4 Pt 1): C840-844Crossref PubMed Google Scholar, 5Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Consequently these interactions are needed for the activity-dependent regulation of contractile force in skeletal muscle (6Sculptoreanu A. Scheuer T. Catterall W.A. Nature. 1993; 364: 240-243Crossref PubMed Scopus (213) Google Scholar) or the β-adrenergic modulation of positive heart inotropism (5Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 7Fink M.A. Zakhary D.R. Mackey J.A. Desnoyer R.W. Apperson-Hansen C. Damron D.S. Bond M. Circ. Res. 2001; 88: 291-297Crossref PubMed Scopus (119) Google Scholar). Furthermore, in heterologous systems, PKA-dependent phos- phorylation of both α1C (CaV1.2) and α1S (CaV1.1) L-type channels does not appear to occur in the absence of AKAP proteins (5Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 8Gray P.C. Johnson B.D. Westenbroek R.E. Hays L.G. Yates III, J.R. Scheuer T. Catterall W.A. Murphy B.J. Neuron. 1998; 20: 1017-1026Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (251) Google Scholar), although the extent of regulation observed in these studies with recombinant systems is much smaller compared with the magnitude recorded in native cells (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar,10Charnet P. Lory P. Bourinet E. Collin T. Nargeot J. Biochimie (Paris). 1995; 77: 957-962Crossref PubMed Scopus (31) Google Scholar). Two distinct membrane-anchored AKAPs (AKAP15/18 and AKAP79/150) have been studied in that context, and a direct interaction between the skeletal muscle L-type channel α1S and AKAP15 has been recently identified (11Hulme J.T. Ahn M. Hauschka S.D. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 30: 30Google Scholar). Whereas AKAP15/18 is prominent in muscles, AKAP79/150 is abundantly expressed in neurons and has a pattern of localization similar to that of α1C (12Hell J.W. Westenbroek R.E. Warner C. Ahlijanian M.K. Prystay W. Gilbert M.M. Snutch T.P. Catterall W.A. J. Cell Biol. 1993; 123: 949-962Crossref PubMed Scopus (648) Google Scholar, 13Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 14Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Cell. 1992; 3: 1215-1228Crossref PubMed Scopus (108) Google Scholar, 15Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (203) Google Scholar). A high proportion of AKAP79/150 and α1C is concentrated in the primary branches of dendrites where they associate with β2-adrenergic receptors (16Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar) to form postsynaptic signaling complexes that regulate synaptic transmission. In the present study, we have examined in heterologous expression models the effect of AKAP79 coexpression on α1C calcium channel activity to further delineate the determinant of their interactions. We found that AKAP79 directly regulates the surface expression of α1C but, surprisingly, independently of PKA activation. A proline-rich region contained within the α1C II-III intracellular linker coordinates this effect. We propose that this sequence, common to various L-type channels, may act as an inhibitory motif that can be masked by AKAP79, favoring the delivery to the plasma membrane of preassembled L-type Ca2+channel signaling complexes. We have previously described the generation of chimeras between α1C and α1E(17Spaetgens R.L. Zamponi G.W. J. Biol. Chem. 1999; 274: 22428-22436Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The II-III loops of α1C and α1E were amplified by PCR and subcloned into c-Myc/pcDNA3. A similar procedure was used to fuse AKAP79 or AKAP79Δ388 with a c-Myc epitope. Deletion of the PP motif on the α1C II-III loop (amino acids 854 to 864) was created by overlapping PCR, using as template a wild-type α1C cDNA engineered to contain two unique silent restriction sites (MluI and SpeI) flanking the II-III loop region (18Stotz S.C. Zamponi G.W. J. Biol. Chem. 2001; 276: 33001-33010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The amplified 780-base pairMluI-SpeI fragment was reintroduced into the template DNA. Accuracy of the different sequences was analyzed by sequencing (Genome Express) and restriction digests. The following cDNA sequences inserted in expression vectors have been used (GenBankTM accession numbers), α1A,M64373; α1C, M67515; α1E, L15453; β1b, NM017346; β2a, M80545; α2-δ1b,AF286488; α1G, AF126965; α1H, NM021098; AKAP79, NM004857. For transient expression in Xenopusoocytes, nuclear injection was performed as previously reported (19Altier C. Spaetgens R.L. Nargeot J. Bourinet E. Zamponi G.W. Neuropharmacology. 2001; 40: 1050-1057Crossref PubMed Scopus (10) Google Scholar). When AKAP79 was coexpressed with the Ca2+ channel subunits, we used a ratio of 1 (AKAP), 3 (Ca2+ channel mix). When needed, this mix was supplemented with the II-III loop constructs at a ratio of 3 (cDNA mix), 1 (II-III loop). As control, the empty vector was used to obtain the same dilution. Oocytes were then incubated at 18 °C for 2–4 days in ND96 medium on rotating platform. For mammalian cell expression, human embryonic kidney (HEK) cells were used and transfected with calcium phosphate as described previously (17Spaetgens R.L. Zamponi G.W. J. Biol. Chem. 1999; 274: 22428-22436Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Macroscopic oocyte currents were recorded using a two-electrode voltage clamp as previously described (19Altier C. Spaetgens R.L. Nargeot J. Bourinet E. Zamponi G.W. Neuropharmacology. 2001; 40: 1050-1057Crossref PubMed Scopus (10) Google Scholar) with 5 mm barium as charge carrier. An AxoPatch-200B amplifier (Axon Instruments) was used for cell-attached recordings with 7–12 MΩ sylgard-coated pipettes filled with a solution containing (in mm): 100 BaCl2, 10 HEPES, pH 7.3. 1 μm FPL64176 was also added to the pipette solution to facilitate the resolution of single channel events. Oocytes were placed in a high potassium solution to reduce the membrane potential to 0 mV. Whole cell recordings in HEK cells were performed with an AxoPatch-200B amplifier using an external solution containing (in mm): 10 BaCl2, 160 TEACl, 10 HEPES (pH to 7.4 with TEAOH). Pipettes of 1–2 MΩ resistance were filled with an internal solution containing (in mm): 110 CsCl, 3 MgCl2, 10 EGTA, 10 HEPES, 3 Mg-ATP, 0.6 GTP (pH to 7.2 with CsOH). All chemicals used were purchased from Sigma except for the peptide Ht-31 provided by Dr. N. Lamb (Montpellier, France). Ht-31 was injected during the recording by an additional microelectrode. pCLAMP7 software was used for data acquisition; analysis was performed with pCLAMP6, Excel, and GraphPad Prism software. Results are presented as the mean ± S.E. and compared using Student’s t test. expression of was using the recently described N. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The hemagglutinin epitope was inserted into the extracellular loop of the epitope with acids to the loop were to form an and into a unique silent at by on the The acid sequence of the the epitope epitope in are in The epitope is acids from the and from the of the Two days cDNA currents were Oocytes were then placed for in ND96 with to incubated for at °C with 1 Molecular in at and incubated for at °C with with for at were with ND96 solution at and then placed in of was in a All data were to the of signal for the channel with and Oocytes protein were as Oocytes were in a containing 100 mm mm (pH 1 mm supplemented with a protein mix were for at °C and at for HEK were as in S.J. S. J. A.M. 2001; PubMed Scopus Google were separated by on or and then The was with proteins were with at proteins were with a at Molecular at or and the were used for When coexpressed with AKAP79, α1C channels a to in This was not with in whole cell with a in the voltage of activation, is following PKA-dependent regulation of native L-type calcium currents α1C β1b, ± α1C AKAP79, ± The of not require the of a calcium channel subunit and ± ± and was not following coexpression of distinct of that the effect of AKAP79 is to the L-type calcium channel coexpression of AKAP79 effect on the of members of the or calcium channel that AKAP79 L-type calcium channels. function of AKAPs is to PKA to the of to their as observed for a number of ionic channels and plasma membrane-anchored AKAPs M. Scott J.D. Cell Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of PKA that was with AKAP79 of L-type channel we that PKA-dependent of of the with the protein kinase not alter the magnitude of the AKAP effect that channel phosphorylation is to the of L-type the of the PKA by an of μm not in functional in α1C channel activity in the or absence of to the interaction is a for the AKAP79 we injected the during the of the recordings with the peptide Ht-31 μm that the and is to of PKA with AKAPs D.W. Stofko-Hahn R.E. Fraser I.D. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). As with or of the Ht-31 peptide not the we created a AKAP79 in the been through of a In both the and the wild-type AKAP79 were to a c-Myc epitope to their with the Ht-31 both and AKAP79 similar in α1C channel activity ± ± these data that the of AKAP79 to L-type calcium channel independently of role in PKA at a novel that be to function as a scaffolding protein Scott J.D. 2000; PubMed Scopus Google Scholar). The in in be to or a of three single channel open probability or an in the number of functional channels. these we examined the effect of the calcium channel is to the the function of AKAP79 were to a effect on then a smaller effect on channels coexpressed with The magnitude of was similar with or AKAP ± ± Although not be for they that AKAP79 does not act in by we performed cell-attached single channel recordings to a AKAP79 effect on A and In the of AKAP79, the number of channels was and the number of channels the channel AKAP79 to act by channel AKAP79 the plasma membrane expression of the channel we directly plasma membrane protein on the described by N. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We inserted the epitope into the extracellular loop of α1C, and to a of the epitope we flanking to the by the M. M. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The channel was by the in and currents were following AKAP79 were to that with the wild-type channels, that the of the additional sequence not channel The to surface expression of an of by using a and a to electrophysiological recordings, were with the primary and surface expression on directly to the of was with a For positive and we used an potassium channel to a signal in this N. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and the α1C As in the L-type channel a signal the is to calcium by an mechanism V. S. E. S. Hoshi T. Mori Y. M. Neuron. 2000; 25: Full Text Full Text PDF PubMed Scopus Google Scholar), we the of the by coexpression of the surface expression of the surface expression of was by coexpression with this not for of the observed in with the role of this subunit in controlling both the expression and the of the subunit C. A. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In coexpression of AKAP79 a further in surface expression of the channel ± that is with the observed of of and surface protein the that AKAP79 to expression of α1C in the plasma We a of calcium channels (17Spaetgens R.L. Zamponi G.W. J. Biol. Chem. 1999; 274: 22428-22436Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, C. Spaetgens R.L. Nargeot J. Bourinet E. Zamponi G.W. Neuropharmacology. 2001; 40: 1050-1057Crossref PubMed Scopus (10) Google Scholar) to the in α1C that in the of We a family of channels that were previously by transmembrane from the α1C L-type channel with from the α1E channel. that that was also contained the intracellular loop II-III loop with As in the of AKAP79 were by of or of α1C with of α1E and of of the AKAP79 A and and the II-III linker as a key cytoplasmic are the for interactions with intracellular we further studied the role of the II-III We examined of the α1C II-III linker or of this loop act to the AKAP79 effect. loops as the region of α1E as a were to a c-Myc epitope in an expression vector and their expression was by from used for electrophysiological were by as as by the same proteins expressed in HEK cells a ratio of channel subunits AKAP79 to II-III the expression of the α1E II-III linker not alter the magnitude of the AKAP79 effect. In the effect of AKAP79 was to in the of the α1C II-III linker II-III This effect to be to the (II-III of the II-III that the of a on II-III and II-III is to the AKAP79 of the α1C II-III loop of the II-III loop expression constructs that were to a to the effect of coexpression of the various II-III loop proteins on the of α1C currents in the of that II-III and II-III act as of the AKAP the expression of the linker The were from to electrophysiological that the various linker proteins have the same expressed in HEK of the acid sequence of the of the α1C II-III we a polyproline motif that is in three of the L-type calcium channel subunits identified to are in we this acid containing the 5 and examined the of AKAP79 to currents by the channel. The not channel the AKAP79 effect the in that were similar to with the channels coexpressed with AKAP79 This effect was not to the oocyte expression an in was the channels were expressed in HEK cells data that the PP region is to channel surface expression and that AKAP79 may to the role of the PP channels not as in the plasma membrane associate with intracellular that are for their to the their and their with to form signal transduction complexes. A number of have been for various Ca2+channel subunits (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar). AKAP proteins are of L-type Ca2+ channel signaling networks (5Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 8Gray P.C. Johnson B.D. Westenbroek R.E. Hays L.G. Yates III, J.R. Scheuer T. Catterall W.A. Murphy B.J. Neuron. 1998; 20: 1017-1026Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 9Fraser I.D. Tavalin S.J. Lester L.B. Langeberg L.K. Westphal A.M. Dean R.A. Marrion N.V. Scott J.D. EMBO J. 1998; 17: 2261-2272Crossref PubMed Scopus (251) Google Scholar, J.T. Ahn M. Hauschka S.D. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 30: 30Google Scholar). The data presented that in to role on kinase and the postsynaptic scaffolding AKAP79 surface expression of L-type Ca2+ channels. is that the surface expression of plasma membrane proteins can be by a number of sequences N. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). include and signals that regulate to the membrane as as that protein from the surface by In we an acid in that the surface Although channels contain of these as the or the signals S. S. J. EMBO J. 2001; 20: PubMed Scopus Google Scholar) are found in the α1C cytoplasmic region identified as for the surface expression from an interaction of different constitutive subunits of a channel as the we are to the of signals in the channels N. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). a role has been described for the calcium channel a signal within the subunit linker V. S. E. S. Hoshi T. Mori Y. M. Neuron. 2000; 25: Full Text Full Text PDF PubMed Scopus Google Scholar). AKAP79 may act on the PP region of channels by a similar although this be in the these data with of studies V. S. E. S. Hoshi T. Mori Y. M. Neuron. 2000; 25: Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. H. Hosey M.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that calcium channels may contain a variety of surface expression that the three a regulation of their cellular We show that three pore-forming subunits contain the PP region in their II-III the of the loop signal is to and channels members of the family have and are not by AKAP79 has been to with a number of the that to the PP we not between AKAP79 and proteins that AKAP79 and α1C and at is that AKAP79 with the L-type calcium channel via or as reported for the interaction M. Dean R.A. Scott Langeberg L.K. R.L. Scott J.D. Neuron. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and molecular studies be to the details of these to the cell mechanism by α1C surface expression is a of is growing evidence that signaling are and data from neurons have that the of AKAP79 with α1C and the β2-adrenergic in AKAPs in signaling to L-type calcium channels via PKA (16Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (444) Google Scholar, M. E. M. and J. W. A. for Scholar). the of this data a novel role of AKAP79 in calcium channel modulation that independently of PKA the same AKAP79 may the of molecular complexes L-type Ca2+ channels and to their to postsynaptic as as calcium In of the role of L-type calcium channels in the of the of gene R.E. U. Science. 2001; PubMed Scopus Google Scholar), a regulation of L-type calcium channel via AKAP proteins may an mechanism for these via controlling L-type channel surface Furthermore, L-type channels, AKAP79/150 proteins have been to gene Y. Rubin C.S. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), the that AKAPs be of the signaling between L-type calcium channels and the that is at postsynaptic sites H. Tsien R.W. Neuron. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). We P. and for wild-type calcium channel subunit and for the subunits, and for the We for and for and for and Lory for of the The provided