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J. Biol. Chem., Vol. 279, Issue 45, 46367-46372, November 5, 2004

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New Short Splice Variants of the Human Cardiac Ca_v2 Subunit

REDEFINING THE MAJOR FUNCTIONAL MOTIFS IMPLEMENTED IN MODULATION OF THE Ca_v1.2 CHANNEL^*

Jo Beth Harry, Evgeny Kobrinsky, Darrell R. Abernethy, and Nikolai M. Soldatov

From the NIA, National Institutes of Health, Baltimore, Maryland 21224

Received for publication, August 18, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Two new short splice variants of the Ca²+ channel ₂ subunit were cloned from human heart poly(A)(+) mRNA. The 410-amino acid _2f subunit is encoded by exons 1A, 2A, 3, 4, 12, 13, and 14 of the human Ca_v2 gene and lacks the protein kinase A phosphorylation site, the -interaction domain (De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495–503), 40% of the -SH3 domain, and 73% of the guanylate kinase domain of the putative membrane-associated guanylate kinases module (McGee, A. W., Nunziato, D. A., Maltez, J. M., Prehoda, K. E., Pitt, G. S., and Bredt, D. S. (2004) Neuron 42, 89–99), and helix 3 of the ₁-subunit binding pocket (Van Petegem F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Nature 429, 671–675). The _2g transcript has two potential initiation codons. With the second ATG codon, it generates the 164-amino acid _2g subunit encoded essentially by the distal part of exon 14, and thus _2g completely lacks any of the above motifs. Immunoprecipitation analysis confirmed stable association of _2f and _2g with the _1C subunit. The plasma membrane localization of _2f and _2g was substantially increased by co-expression of the _1C,77 and ₂ subunits. In COS1 cells, _2f and _2g increased plasma membrane targeting of the pore-forming _1C subunit and differentially facilitated (_2f > _2g) the voltage gating of otherwise silent Ca_v1.2 channels. We conclude that it is unlikely that the -interaction domain, membrane-associated guanylate kinases module, and the ₁-subunit binding pocket helix 3 are essential for the interaction of the _1C and ₂ subunits and suggest that in addition to the ₁-subunit binding pocket helices 5 and 8, a yet unresolved C-terminal ₂ region plays a crucial role.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The L-type high voltage-activated Ca_v1.2 channel is composed of the pore-forming _1C subunit and the auxiliary and ₂ subunits. The Ca_v subunits (1) are essential cytoplasmic modulators of the Ca²⁺ channel activity that generate a molecular signal necessary for the facilitation of voltage gating as well as for the correct plasma membrane targeting of the functional Ca_v1.2 complex (2). The _1C and subunits are tightly associated in the channel complex and can be co-immunoprecipitated in mild non-ionic detergents. A conserved -interaction domain (BID)¹ common for genetically different subunits (₁–₄) was proposed as a binding motif interacting with the conserved -interaction domain (AID) of the I-II cytoplasmic linker between repeats I and II of an ₁ subunit (3, 4). The ₁- interaction is believed to chaperon the channel by inhibiting an endoplasmic reticulum retention signal encoded in the ₁ subunit I-II linker (5). Comparative studies showed that subunits differentially modulate inactivation kinetics (6) and single-channel properties of the Ca_v1.2 channel (7). We have found that the differential -subunit modulation predominantly influences the slow inactivation of the channel and is associated with distinct voltage-gated rearrangements between the N-terminal regions of the _1C and ₂ subunits (8). Structural principles underlying the -subunit modulation of Ca²⁺ channels were also approached by a search of structural homology with known regulatory proteins. It has been found (9) that the vast central conservative region of ₂ subunits shares distant homology with the Src homology 3 (SH3)-guanylate kinase (GK) module of membrane-associated guanylate kinases (MAGUKs). This hypothesis was further elaborated by studying mutations of the rat (N terminus-palmitoylated) _2a subunit that interfere with interactions between SH3 and GK and affect inactivation of Ba²⁺ currents (10, 11). Intramolecular interactions between the -subunit SH3 and GK homologues were supported by the results of the recent high-resolution crystallography studies of the -subunit "cores" of SH3-GK domains alone or in complex with AID (12–14). These studies also showed that BID is not available for the binding to AID because it is buried inside the -subunit structure. Instead, the -subunit binding pocket (ABP) distal to the SH3 domain was inferred as a structure engaged in the interaction with AID.

The first cardiac ₂ subunits cloned from rat (15) and rabbit (16) hearts have over time been co-identified with five N-terminal splice variants (_2a–_2e; for overview, see Ref. 17). A recent comprehensive study revealed that in the human left ventricle there are nine Ca_v2 splice variants, including _2b, _2c, _2d, and _2e; the exon 7C isoforms of _2b, _2c, and _2d; the exon 7B isoform of _2b; as well as the _2b transcript lacking exon 7 and truncated in the exon 8 region (18). Here, we report on two new splice variants of the Ca²⁺ channel ₂ subunit gene cloned from the human normal heart poly(A)(+) mRNA and sequentially named _2f and _2g. The _2f and _2g transcripts lack exons encoding a single protein kinase A (PKA) phosphorylation site, BID, as well as a large part (_2f) or the entire SH3-GK module (_2g). Despite the wide structural differences between _2f/_2g and the rest of the ₂ subunits, the new ₂ subunits yielded fully functional Ca_v1.2 channels with different inactivation characteristics when co-expressed in COS1 cells with the ₂ and the human vascular _1C,77 subunits. These data indicate the need to redefine the significance of the previously outlined subunit functional motifs and implicate the C-terminal region encoded by the distal ₂ gene exon in modulation of the channel.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cloning of the Short Human Cardiac Ca_v2 Subunits—The Ca²⁺ channel _2f and _2g subunits were cloned from the human heart poly(A)(+) mRNA pooled from three Caucasian males (ages 35, 55, and 55; cause of death, trauma) (BD Biosciences Clontech, Palo Alto, CA; catalog number 6533-1) using C. therm. Polymerase One-Step reverse transcriptase (RT)-PCR (Roche Applied Science). The RT reaction was carried out for 30 min at 57 °C with a common RT oligonucleotide primer, 5'-ACATATGATTGCAGTGTAGACC-3', designed from the ₂ subunit 3'-untranslated region (nt 804058–804079 in the Human Genome Contig NT_8705.15 of chromosome 10). In the first round of PCR, a common antisense 5'-GCTGTTAGTTATACAAGACTTC-3' primer (nt 804014–804035) was used to amplify _2f and _2g with the sense _2d-specific 5'-ATGGTCCAAAGGGACATGTC-3' (nt 404991–405010) or the _2b-specific 5'-ATGCTTGACAGACGCCTTATA-3' (nt 605181–605201) primer, respectively. PCR mixtures were denatured for 2 min at 94 °C followed by 30 PCR cycles, each composed of denaturing for 45 s at 94 °C, annealing for 1 min at 51 °C, and extension for 2.5 min at 72 °C, with the last step of the last cycle extended to 10 min. For the second round of PCR with "nested primers," a common antisense 5'-gctctagagTCATTGGCGGATGTAAACATC-3' primer (nt 803958–803978, attached to an XbaI linker shown in small letters) was used with the sense 5'-gccaccATGGTCCAAAGGGACATGTCCAAG-3' (_2f) and 5'-gccaccATGCTTGACAGACGCCTTATAGCTC-3' (_2g) primers (incorporating Kozak sequence, shown in small letters in front of the ATG start codon) and the same PCR conditions except with an increased annealing temperature (58 °C). The RT-PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA), ligated into the pCR2.1 TA vector (Invitrogen) and sequenced in both directions. To generate _2g, the _2g-coding TA clone was amplified using an antisense primer, as shown above for the second round of PCR, and the sense primer 5'-cgggatccgccaccATGTATCTCTGGAGGAGGACCGGG-3', which starts at the second ATG codon and has a Kozak sequence preceded by a BamHI linker at the 5'-end. For expression in eukaryotic cells, the _2f- and _2g-coding clones were ligated into the pcDNA3 vector at the XbaI (filled)–NotI and BamHI–XhoI sites, respectively. To systematically investigate subcellular localization of the _1C and subunits in situ, their N termini were genetically fused to EYFP and ECFP, respectively. Experimentally, such labeling did not interfere with the functional expression of the channel in our previous (8, 19) and current investigations as compared with the unlabeled subunits. To generate (ECFP)_N-_2f, the _2f coding sequence was supplemented with the 5'-NcoI (Klenow-blunted) and 3'-ApaI linkers and ligated into the 5'-ECFP-pcDNA3 vector at the XhoI (filled) and ApaI sites. To generate (ECFP)_N-_2g, the _2g-coding sequence was supplemented with the 5'-BamHI and 3'-XhoI linkers and ligated into the 5'-ECFP-pcDNA3 vector at the respective sites.

Immunoprecipitation Analysis—The (ECFP)_N-labeled _2f or _2g subunits (replaced by EGFP in the negative control) were expressed in HEK293 cells in the presence or in the absence of _1C,77 and ₂. The labeled proteins were co-immunoprecipitated with Living Color full-length A.V. polyclonal antibody (BD Biosciences Clontech) as described in detail earlier (8). The (ECFP)_N-labeled subunits were identified on Western blots by Living Color monoclonal antibody JL-8 (1:8,000 dilution; BD Biosciences Clontech). The co-precipitated _1C subunits were detected with the affinity-purified rabbit anti-_1C calcium channel polyclonal antibody (1:10,000; Chemicon International) using an ECL Plus Western blotting detection system (Amersham Pharmacia Biotech).

Electrophysiology—Expression in COS1 cells, all electrophysiological measurements, and fluorescence microscopy were carried out under standard conditions described in Kobrinsky et al. (8) and thus allow for direct comparison with our previous investigations of the _1a and ₂ subunits. Briefly, COS1 cells were plated on poly-D-lysine-coated coverslips (MatTek) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected with cDNAs coding for the _1C, , and ₂ subunits (1:1:1, weight for weight) using the Effectene kit (Qiagen). Membrane currents in transfected COS1 cells were recorded 48–72 h after transfection by the patch clamp technique in the whole-cell configuration from individual single cells. The extracellular (bath) solution contained (in mM) 100 NaCl, 20 BaCl₂ or CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES at pH 7.4. The internal (pipette) solution contained (in mM) 110 CsCl, 5 MgATP, 10 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate, 20 tetraethylammonium, 0.2 cAMP, and 20 HEPES at pH 7.4. When filled with the internal solution, pipettes (Kimax-51, Kimble, Vineland, NJ) had resistance ranging between 3 and 6 megohms. Current tracings were low pass-filtered at 1 kHz and sampled at 2.5–5 kHz. Data were acquired using the Axopatch 200B amplifier and pClamp 8.1 software (Axon Instruments, Union City, CA) and corrected for leakage using an on-line P/4 subtraction paradigm.

Fluorescent Microscopy—All cell images were obtained on an epifluorescent Nikon microscope TE200 equipped with a 10-bit digital Hamamatsu CCD camera C4742–95 (Hamamatsu, Bridgewater, NJ). Image processing and analysis were performed with the SimplePCI 5.3 software (Compix, Pittsburgh, PA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Molecular Cloning and Structural Properties of the Short Human Cardiac _2f and _2g Subunits—Two new short splice variants of the Ca²⁺ channel ₂ subunit gene, named _2f and _2g (Fig. 1A), were cloned by RT-PCR from the poly(A)(+) fraction of the human normal heart mRNA. The RT and PCR primers for the cloning were deduced from the Human Genome Project draft sequence NT_008705 [GenBank] .15 of chromosome 10. The _2f subunit has a theoretical molecular mass of 45.8 kDa, is composed of 410 aa, and has the N terminus encoded by exons 1A and 2A analogous to _2d (18). However, missing in the _2f transcript are exons 5–11 coding for the 250-aa central region of the ₂ protein, including BID, as well as 55 of 136 aa (i.e. 40%) of the C-terminal part of the SH3 domain and 192 of 262 aa (i.e. 73%) of the N-terminal part of GK domain. Overall, the following crystallographically resolved structures (13) are absent from the _2f protein: antiparallel -strands 3–5 and the second -helix (2) of the first conserved domain, the entire variable domain V2, as well as parallel -sheets 6–9, two -helices (3–4), and two 3₁₀ helices (2-3) of the second conserved domain. Thus, the entire linker between SH3 and GK ("HOOK domain") present in MAGUKs is deleted from the _2f subunit.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Cloning and co-immunoprecipitation analysis of the 2f and 2g subunits. A, a schematic map of the human cardiac 2 subunit transcripts including all exons. The 2f and 2g transcripts are compared with 2d. Exons (boxed) are numbered according to Foell et al. (18), and their size (in base pairs) is shown below each exon. ATG and TGA above the arrows indicate initiation and termination codons, respectively. An approximate position of BID (bold horizontal line) and SH3 and GK motifs (lines with double arrowhead) is illustrated above the 2d transcript. The spliced-out exons in the 2f and 2g transcripts and their parts in exons 3 and 14 of the 2g transcript are shown as gray boxes (unnumbered). Note that the 2g transcript is initiated from exon 2c similar to 2b. Because the reading frame of the 2g transcript opened at the first initiation codon is interrupted at the position 153, the second ATG (shown above exon 3) at the position 98 was utilized to generate the 2g transcript. B, co-immunoprecipitation with the short splice variants. The (ECFP)N-2f(1), (ECFP)N-2g (2) or no subunit (3,4) was co-expressed in HEK293 cells with (1–3) or without 4) the 1C,77 and 2 subunits. EGFP was co-expressed in control sample 3. Cells were lysed by freeze-thaw. To remove free subunits and other cytosolic proteins, the crude membrane particulate fractions were prepared from the lysed cells as described earlier (8). Membranes were solubilized with 0.5% Nonidet P-40, and the channel proteins were immunoprecipitated with the antibody against green fluorescent protein, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting. The (ECFP)N-labeled 2 splice variants (1,2) and EGFP (3) were detected on the blot by the monoclonal antibody against green fluorescent protein, whereas the co-immunoprecipitated 1C subunit was detected using the anti-1C antibody.

2g

2g

2g

Although _2f and _2g were cloned from the polyadenylated fraction of mRNA, none of the studied short ₂ splice variants has yet been shown to be expressed in the human heart as functional proteins. Because of this and as the usage of initiation codons in _2g remains unknown, _2g is considered a putative splice variant of the _2g subunit. Nevertheless, because _2g lacks not only BID and the entire SH3-GK domains but also the variable ₂-subunit N termini-coding exons, _2g is particularly interesting for the study of the functional significance of these genetically deleted motifs. To investigate whether the structural deletions discovered in the new ₂ splice variants affect their interaction with the _1C subunits, co-immunoprecipitation of the short ₂ subunits with the wild-type _1C,77 was analyzed by Western blot assay (Fig. 1B). ECFP was genetically fused to the N termini of the new ₂ splice variants by in-frame ligation of the ECFP and _2f/_2g coding sequences, and the (ECFP)_N-labeled ₂ subunits were co-expressed in HEK293 cells with the ₂ and _1C,77 subunits. Similar to the _1a and the _2(r) subunits (8), the (ECFP)_N labeling did not compromise their functional activity (data not shown). Use of antibody against GFP to co-immunoprecipitate (ECFP)_N-₂ variants and _1C,77 from the solubilized membrane particulate fraction of HEK293 cells helped to avoid a contamination with the nonassociated cytosolic (ECFP)_N-₂ and the membrane-bound orphan _1C subunits. Both _2f (Fig. 1B, lane 1) and _2g (lane 2) pulled down the _1C,77 protein from the solubilized membrane preparations suggesting stable association between the subunits. A similar observation was made with _1a, _2(r) (8), and other proteins. In controls, no immunoprecipitation of the _1C subunit by anti-GFP antibody was found in the absence of (ECFP)_N-₂ variants in COS1 cell with (Fig. 1B, lane 3) or without (lane 4) EGFP co-expressed. These data indicate that the ₂ and _1C subunits are necessary for the plasma membrane targeting by the _2f and _2g subunits.

The _2f Subunit Stimulates the Plasma Membrane Targeting and Facilitates the Ca_v1.2 Channel Voltage Gating—To better characterize the cellular location of the expressed _1C and subunits, the (ECFP)_N-labeled _2f and _2g were expressed with _1C,77 and ₂ subunits in COS1 cells lacking the endogenous Ca²⁺ channels (21). In contrast to the data obtained with Xenopus oocytes (10, 14) and HEK293 cells (11), heterologous expression of the _1C and ₂ subunits in COS1 cells did not induce an appreciable Ca²⁺ channel activity (Fig. 2A) unless a subunit was co-expressed (Fig. 2B). Thus, selection of the COS1 cells expression system allowed us to avoid ambiguity in the assessment of the -subunit modulation of the Ca²⁺ channel typical for the cited studies (10, 11, 14) and to define the -subunit modulation here as a facilitation of the Ca²⁺ channel voltage gating by a subunit. Essential prerequisites for a -subunit modulation of the channel are binding of Ca_v to the _1C subunit and targeting of the oligomeric channel complex to the plasma membrane.

View larger version (55K): [in this window] [in a new window]   FIG. 2. Effect of the 2f subunit on functional expression, subcellular distribution, and electrophysiological properties of the Ca2+ channel. A, IBa recorded from a COS1 cell transfected by the (EYFP)N-1C,77 and 2 subunits (inset shows the whole-cell EYFP) in response to a 600-ms test pulse to +20 mV from the holding potential of –90 mV. B, panel a, representative traces of IBa in response to the indicated test pulses (Vh = –90 mV) from a COS1 cell transiently transfected with the (EYFP)N-1C,77, 2 and 2f subunits. Panel b, phase-contrast cell image with a shadow of patch pipette; panel c, whole-cell EYFP fluorescence. Panel d, average normalized I-V relationship (n = 9) for IBa through the (EYFP)N-1C,77/2/2f channel. C, whole-cell fluorescence from COS1 cells expressing the (EYFP)N-1C,77 and 2 subunits in the absence (a) or in the presence (b) of the 2f subunit or from COS1 cells expressing the (ECFP)N-2f and 2 subunits in the absence (c) or in the presence (d) of the 1C,77 subunit. The expression level of the labeled proteins was approximately the same (compare unadjusted fluorescence scales in the right panels). D, representative traces of ICa (a) or IBa (b–d) evoked by a test pulse to +20 mV applied for 0.6 (a, b), 5 (c), or 30 s (d). E, whole-cell fluorescence from COS1 cells expressing the (ECFP)N-2(r) subunit in the absence (a) or in the presence (b) of the 1C,77 and 2 subunits. Control panel c shows surface membrane targeting of (EYFP)N-1C,77 co-expressed with the 2 and 2(r) subunits. Panel d, representative 30-s trace of IBa through the (EYFP)N-1C,77/2/2(r) channel (8). Vh = –90 mV. Dashed lines show a zero current. Scaling bars, 3 µm.

Despite the large structural differences between _2f and the other known ₂ subunits, when co-expressed in COS1 cells with the human vascular _1C,77 subunit and ₂, the new _2f subunit yields a fully functional Ca_v1.2 channel. Fig. 2B (panel c) shows representative traces of the Ba²⁺ current (I_Ba) evoked by the 600-ms test pulses to the indicated voltages applied from V_h = –90 mV. The corresponding averaged normalized I-V relationship is presented in Fig. 2B, d. The values for the half-maximal activation (V_0.5 = 1.8 ± 1.7 mV) and the slope factor (k_I-V = –7.9 ± 0.9; n = 9) were essentially similar to those reported earlier for I_Ba through the _1C,77/₂/_2(r) channel expressed in Xenopus oocytes (22). Respectively, a replacement of Ba²⁺ for Ca²⁺ as the charge carrier (Fig. 2D, a) produced a typical strong acceleration of the current decay characteristic for Ca²⁺-induced inactivation of the Ca_v1.2 channel. Contrasting with the rabbit _2(r) subunit, the new short _2f renders notably faster inactivation of I_Ba. Fig. 2D (b–d) shows maximum I_Ba elicited by test pulses of different durations to +20 mV. A two-exponential fitting of the 30-s I_Ba through the (EYFP)_N-_1C,77/₂/_2f channel (n = 5) showed the inactivation time constants () and fractions (I) of the fast and slow components to be _f = 465 ± 20 ms (I_f = 46.4 ± 1.4%) and _s = 4.31 ± 0.68 s (I_s = 35.2 ± 2.1%), respectively. Although complete inactivation of the current was not reached even with the 45-s pulse, the fraction of the current that remains by the end of the 30-s pulse (18.0 ± 2.0%) was, on average, significantly (p < 0.05) smaller that those we have found (27.5 ± 3.3%) with the _2(r) subunit. The fast component of I_Ba through the (EYFP)_N-_1C,77/₂/_2(r) channel (Fig. 2E, d) (_f = 499 ± 68 ms; I_f = 53.4 ± 1.6%; n = 5) was essentially similar to those of the _2f channel current. However, the slow component showed a delayed decay (_s = 12.88 ± 2.40 s; I_s = 19.4 ± 4.7%; p < 0.01). Overall, our study revealed that _2f exhibits properties characteristic of the ₂ subunits and yields a fully functional Ca_v1.2 channel. These data suggest that a requirement of the subunit for functional conformation of the channel (6, 23) is conserved in the regions of ₂ other than BID and the missing essential parts of SH3-GK.

The _2g Subunit Narrows the Functional Correlates of the Subunit to the C-terminal 153-Amino Acid Region—The immunoprecipitation analysis (Fig. 1B, lane 2) showed that the _2g subunit binds to _1C,77 despite the lack of BID, the entire MAGUK module, and the ABP. The _2g subunit is composed of 164 aa of which the N-terminal 11 residues are new to ₂ subunits. The last 153 aa are common for the C-terminal region of ₂ subunits encoded by the distal part of exon 14. Our data indicate that this C-terminal region is sufficient to confer the assembly of the functional channel. Fig. 3 shows cellular distribution and functional expression of the Ca²⁺ channel assembled with _2g. When co-expressed with ₂ but in the absence of _1C, the (ECFP)_N-_2g subunit was diffusely distributed over the cytoplasm without selectively targeting the plasma membrane (Fig. 3A, a). The _1C,77 subunit strongly enhanced accumulation of (ECFP)_N-_2g in the plasma membrane (Fig. 3A, panel b). Conversely, _2g effectively increased membrane targeting of the (EYFP)_N-labeled _1C,77 (Fig. 3A, compare c and d). Thus, _2g retains the molecular signals necessary for binding to the _1C subunit and plasma membrane targeting of the channel.

View larger version (53K): [in this window] [in a new window]   FIG. 3. Cellular distribution and functional expression of the Ca2+ channel directed by 2g in COS1 cells. A, comparison of whole-cell fluorescence from COS1 cells expressing the (ECFP)N-2g and 2 subunits in the absence (a) or in the presence (b) of the 1C,77 subunit or from COS1 cells expressing the (EYFP)N-1C,77 and 2 subunits in the absence (c) or in the presence (d) of the 2g subunit. Right panels show fluorescence scales. B, representative traces of ICa (left) and IBa (right) through the (EYFP)N-1C,77/2/2g channel evoked by 600-ms test pulses to +20 mV from Vh = –90 mV. C, representative 30-s trace of IBa recorded under the same voltage protocol. Dashed line shows zero current. Scaling bars, 3 µm.

2g

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	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The most interesting result of this study is that vast deletions in the central region of the ₂ subunit that include most of or the entire SH3-GK region do not compromise the -subunit modulation of the Ca²⁺ channel. The _2f subunit exhibits an array of properties that resemble those of the _2(r) and other ₂ subunits, whereas the shortest known _2g isoform shows more unusual features in modulation of the channel. Our results with the _2f subunit confirm the main structural implications of the crystallographic study of the Ca_v2a-AID complex (12–14) by highlighting the fact that it is the C-terminal region encoded by exons 12–14 that appears to bear essential structures involved in interaction with an ₁ subunit. Although it is not clear to what extent the identified structural features of the ₂"conserved core"are preserved in its remnants in the _2f subunit, they may include the C-terminal fragment of the helix 5 (153–159) and the entire helix 8 (196–215) that jointly form a part of ABP (13). However, _2f lacks the helix 3 co-identified with the ABP. It is unlikely that the small parts of the SH3 and GK domains remaining in the _2f subunit are sufficient to support their cross-interaction, because the distal loop and strand E of the conserved domain I implemented in this interaction (12) are both absent from _2f.

The remnant of the first conserved domain of _2f, encoded in part by exons 3 and 4 (aa 73–152), shows 72–80% homology with the other cloned human subunits, including _1B-1 and _1B-2 (24), ₃ (25), and ₄ (26). The remnant sequence (aa 153–217) of the second conserved region of _2f, which includes helices 5 and 8 of the ABP encoded in part by exons 12 and 13, shares 83–88% of the overall homology with other subunits. Experimentally established conventional modulation of the channel by the _2f subunit suggests that the combination of 5 and 8 helices is an important structural requirement for ABP and/or that helix 3 might be replaced in ABP by an unidentified motif(s) in _2f. In contrast, _2g lacks any structural bases for ABP or MAGUK, and _2g does not share a substantial (>30%) homology with the ₁, _3, and ₄ subunits. Nevertheless, _2g binds to the _1C,77 subunit (Fig. 1B, lane 2), stimulates membrane targeting of the channel (Fig. 3), and shows properties of a weak modulator of the Ca_v1.2 channel voltage gating. Specifically, _2g supports slowly inactivating Ba²⁺ currents at prolonged depolarization, which is a characteristic feature of other ₂ subunits. There are no structural data available for the ₂-subunit C-terminal region to explain the results of the functional evaluation of the _2g except to suggest that within the C-terminal 153-amino acid sequence, an essential structure of the ₂ subunit is present that endows a modulation of the channel via functional interaction with _1C.

The subunit is an important component of a molecular complex that determines cytoplasmic helical packing stabilizing channel gating. We hypothesize that it is the Ca_v2 N-terminal half that finely tunes the gating facilitation and affects differential modulation of the Ca²⁺ channel current inactivation and the voltage-gated rearrangements of the _1C and subunit N-tails (8). The MAGUK module may contribute to this regulation, but its partial or even complete deletion does not impede the channel modulation as defined in this paper.

Several Ca_v variants with truncated C-terminals were previously identified (18). Our work has established the fact that the main splice isoforms (_2a–_2e) of the ₂ subunit gene may generate smaller functional variants through the genetically encoded deletions of the central regions. These data interject significant complications in the reassessment of tissue and cellular distribution of Ca_v2 variants. Development of specific immunohistochemical tools for _2f and _2g is problematic because they share the same aa sequences with other ₂ subunits. Given the large size of untranslated regions of the ₂-subunit transcripts (15), relatively small structural deletions encoded in _2f and _2g may be difficult to assess by Northern blot analysis.

Our results also show that alternative splicing of the ₂ subunit gene may affect regulation of the Ca_v1.2 channels in the human heart by PKA. Activation of PKA through the -adrenergic receptor pathway is crucial for an up-regulation of the cardiac L-type Ca²⁺ currents (27). This effect was found to be in part because of PKA phosphorylation of a single specific site of the ₂ subunit (20). Because this site is genetically deleted from the naturally occurring short splice variants of the human ₂ subunit described in this paper, these variants may have a distinct role (or no role) in human cardiac electrophysiology. In any case, the discovery of the new functional short ₂-subunit isoforms lacking many of the predicted functional motifs adds understanding to the molecular bases for the -subunit modulation of Ca²⁺ channels and may give rise to new molecular tools of the study of mechanisms of Ca²⁺ signal transduction.

	FOOTNOTES

 
^* This work was supported by the Intramural Research Program, NIA, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY675091 [GenBank] (_2f) and AY675092 [GenBank] (_2g).

Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 410-558-8343; Fax: 410-558-8318; E-mail: soldatovN{at}grc.nia.nih.gov.

¹ The abbreviations used are: BID, -interaction domain; AID, -interaction domain; SH3, Src homology 3; GK, guanylate kinase domain; MAGUK, membrane-associated guanylate kinase; ABP, -subunit binding pocket; PKA, protein kinase A; RT, reverse transcriptase; nt, nucleotide(s); EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein; aa, amino acid(s); HEK, human embryonic kidney; GFP, green fluorescent protein; _2(r), rabbit cardiac ₂ subunit; V_h, holding potential.

	ACKNOWLEDGMENTS

 
We are grateful to Mike Shi for help with electrophysiology and to Sarah Bentil and Yasir Kazmi for help with fluorescent images.

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