Characterization of C-terminal Splice Variants of Cav1.4 Ca2+ Channels in Human Retina*

Voltage-gated Ca2+ channels (Cav) undergo extensive alternative splicing that greatly enhances their functional diversity in excitable cells. Here, we characterized novel splice variants of the cytoplasmic C-terminal domain of Cav1.4 Ca2+ channels that regulate neurotransmitter release in photoreceptors in the retina. These variants lack a portion of exon 45 and/or the entire exon 47 (Cav1.4Δex p45, Cav1.4Δex 47, Cav1.4Δex p45,47) and are expressed in the retina of primates but not mice. Although the electrophysiological properties of Cav1.4Δex p45 are similar to those of full-length channels (Cav1.4FL), skipping of exon 47 dramatically alters Cav1.4 function. Deletion of exon 47 removes part of a C-terminal automodulatory domain (CTM) previously shown to suppress Ca2+-dependent inactivation (CDI) and to cause a positive shift in the voltage dependence of channel activation. Exon 47 is crucial for these effects of the CTM because variants lacking this exon show intense CDI and activate at more hyperpolarized voltages than Cav1.4FL. The robust CDI of Cav1.4Δex 47 is suppressed by CaBP4, a regulator of Cav1.4 channels in photoreceptors. Although CaBP4 enhances activation of Cav1.4FL, Cav1.4Δex 47 shows similar voltage-dependent activation in the presence and absence of CaBP4. We conclude that exon 47 encodes structural determinants that regulate CDI and voltage-dependent activation of Cav1.4, and is necessary for modulation of channel activation by CaBP4.

In the retina, voltage-gated Ca v 1.4 (L-type) Ca 2ϩ channels are localized in the synaptic terminals of rod and cone photoreceptors where they mediate Ca 2ϩ signals that trigger glutamate release at the first synapse in the visual pathway (1). In mice lacking expression of Ca v 1.4, there is a complete loss of photoreceptor synaptic transmission and a failure in photoreceptor synapse maturation (2)(3)(4)(5)(6). The importance of Ca v 1.4 for vision in humans is illustrated by the disorders associated with mutations in the CACNA1F gene encoding the pore-forming ␣ 1 subunit of Ca v 1.4. These include congenital stationary night blindness type 2 (CSNB2 (7)), 2 X-linked cone-rod dystrophy (8,9), and Åland island eye disease (10).
Compared with Ca v 1.2 channels that are prominent in the brain and heart, Ca v 1.4 channels activate at more negative voltages, and show very little inactivation during sustained depolarizations (11)(12)(13). It is thought that these properties support tonic glutamate release at the membrane potential of photoreceptors in darkness (Ϫ30 to Ϫ40 mV (14,15)). Unlike other Ca v channels, Ca v 1.4 does not undergo Ca 2ϩ -dependent inactivation (CDI) (11)(12)(13)), a negative feedback regulation by incoming Ca 2ϩ ions. For Ca v 1 and Ca v 2 channels, CDI is mediated by calmodulin (CaM) binding to site(s) in the proximal C-terminal domain of the pore-forming ␣ 1 subunit (reviewed in Refs. 16 and 17)). In Ca v 1.4, a sequence in the distal C-terminal domain (C-terminal automodulatory domain, CTM) suppresses CDI through an intramolecular interaction with the proximal C-terminal domain (18 -20). In addition to effects on CDI, the CTM inhibits voltage-dependent activation of Ca v 1.4. A CSNB2causing mutation deletes the CTM from Ca v 1.4 (K1591X (21)), unmasks strong CDI, and causes a hyperpolarizing shift in the voltage dependence of activation (22), both of which would be expected to decrease the dynamic range of photoreceptor signal transmission (15).
Like other Ca v channels, Ca v 1.4 undergoes alternative splicing that can greatly alter the functional properties of the channel. For example, a splice variant that removes a large fraction of the C-terminal domain including the CTM (Ca v 1.4 ex43*) is expressed in human retina and exhibits robust CDI and hyperpolarized activation voltages in transfected HEK-293 cells (23). Such properties, as in K1591X (22), might be expected to cause pathological changes in visual signaling. However, photoreceptors in the retina express CaBP4, a member of a family of Ca 2ϩbinding proteins (CaBPs) related to CaM (24). CaBP family members prevent CDI of Ca v 1 channels (25)(26)(27), in part by competing with CaM for binding sites on the channel (28 -30). CaBP4 binds to the C-terminal domain of Ca v 1.4 channels containing the CTM, and enhances voltage-dependent activation. CaBP4 does not affect CDI, which is already nullified in fulllength Ca v 1.4 channels (31,32). Coexpression of CaBP4 with Ca v 1.4 channels lacking the CTM strongly suppresses CDI as in full-length channels (32). Therefore, splice variants lacking the CTM may exhibit properties consistent with native photoreceptor Ca v channels (i.e. no CDI (33)) in contrast to their properties in transfected HEK-293 cells (23).
Electrophysiological analysis of Ca v 1.4 is challenged by the small current densities produced by these channels in heterologous expression systems. One strategy to overcome this hurdle is to fuse a portion of Ca v 1.4 (e.g. the CTM) to the core of Ca v 1.2 or Ca v 1.3 channels, giving rise to more robust currents (23,34). Another caveat is that virtually all studies to date have utilized auxiliary Ca v ␤ and ␣ 2 ␦ subunits that may not be associated with native Ca v 1.4 channel complexes in photoreceptors. Of the 4 Ca v ␤ and ␣ 2 ␦ variants that have been characterized (35,36), ␤ 2 and ␣ 2 ␦ 4 are required for vision in mice (37)(38)(39). We previously showed that most photoreceptor Ca v 1.4 channels contain an unusual ␤ 2 splice variant (␤ 2ϫ13 ) as well as ␣ 2 ␦ 4 (40). To gain insights into how alternative splicing affects Ca v 1.4 channels containing ␤ 2ϫ13 and ␣ 2 ␦ 4 , we analyzed the electrophysiological properties of new Ca v 1.4 splice variants that we discovered while isolating cDNAs encoding Ca v 1.4 from human retina. Unlike Ca v 1.4 ex43*, which lacks the C-terminal 256 amino acids of the channel (23), these variants lack only exon 47, which deletes part of the CTM, but leaves the remaining C-terminal domain intact. We show that these variants are expressed at significant levels in human retina, and exhibit hyperpolarized voltages of activation and CDI similar to Ca v 1.4 ex43*. CaBP4 binds to and inhibits CDI of channels lacking exon 47, although it does not further enhance the hyperpolarized voltage-dependent activation of these channels. We conclude that exon 47 encodes critical determinants for regulating CDI and activation in a heterologous expression system, but that the presence of CaBP4 would likely nullify the CDI while not affecting the activation properties of these variants in vivo. Our results highlight the importance of analyzing Ca v channels in the presence of known modulators for understanding the impact of alternative splicing on the properties of the native channels.  Fig. 2A). The inability to measure Ca v 1.4⌬ex p45,47 in mouse retina could be due to expression of Ca v 1.4⌬ex p45,47 primarily in cone photoreceptors, which are more abundant in the retina of primates than mice. To test this, we compared the expression of Ca v 1.4 FL and Ca v 1.4⌬ex p45,47 in the cone-rich macula of monkey retina. No significant difference was observed between the ratio of Ca v 1.4 FL and Ca v 1.4⌬ex p45,47 in the macula compared with the peripheral retina of the monkey (Fig. 2, C and D). Therefore, Ca v 1.4⌬ex p45,47 is unlikely to be more enriched in cones compared with rods. Quantitative PCR revealed that Ca v 1.4⌬ex p45,47 was highly expressed in human and monkey retina, albeit at 20 -150 lower levels than Ca v 1.4 FL (Fig. 2, B-D; see Table 1 for primer sequences). In additional experiments, we detected Ca v 1.4 transcripts that had the single partial deletion of exon 45 (Ca v 1.4⌬ex p45) or full deletion of exon 47 (Ca v 1.4⌬ex47). Of these, Ca v 1.4⌬ex p45 was the most abundant ( Fig. 3, A-C).

Identification of Novel
Ca v 1.4 Variants Lacking Exon 47 Exhibit Robust CDI-Although C-terminal splice variants including Ca v 1.4⌬ex p45 have been characterized (23), those lacking exon 47 have not. Because deletion of exon 47 removes part of the CTM (Fig. 1,  A and B), we predicted that its deletion might affect CDI and voltage-dependent activation. We tested this in whole cell patch clamp recordings of HEK293T cells transfected with Ca v 1.4 FL , Ca v 1.4⌬ex p45, Ca v 1.4⌬ex47, or Ca v 1.4⌬ex p45,47. Cells were cotransfected with cDNAs encoding the auxiliary ␤ 2ϫ13 and ␣ 2 ␦ 4 subunits that co-assemble with Ca v 1.4 in the retina (40). To study CDI, we compared inactivation of Ca 2ϩ currents (I Ca ) with that of Ba 2ϩ currents (I Ba ). Inactivation was measured as the residual current amplitude at the end of the pulse normalized to the peak current amplitude (Fractional I); CDI was calculated as F CDI (difference in Fractional I Ca and mean Fractional I Ba at Ϫ20 mV).
As shown previously, Ca v 1.4 FL currents showed little inactivation during 1-s depolarizations regardless of whether Ca 2ϩ or  Fig. 5, A-C). To more rigorously characterize the voltage dependence of channel activation, we plotted the normalized tail current amplitudes against test voltage (Fig. 5, D-F). Boltzmann fits of the data indicated a significant effect of exon 47 deletion on the half-maximal voltage (V h ) and slope (k). The V h was significantly more negative and k was steeper for Ca v 1.4⌬ex47 and Ca v 1.4⌬ex p45,47 than for Ca v 1.4 FL (Fig. 5, E and F; Table 3). In contrast, there was no significant difference in these parameters for Ca v 1.4⌬ex p45 and Ca v 1.4 FL ( Fig. 5D; Table 3 6A). As we have found previously for FLAG-tagged Ca v 1.3 (42), channel protein was detected by Western blotting only after immunoprecipitation and not in the cell lysates presumably due to limited sensitivity of the FLAG antibodies. These co-immunoprecipitated proteins were not detected when control mouse IgG was used instead of anti-FLAG antibodies. Although these results suggested that CaBP4 binds equally well to both Ca v 1.4 and Ca v 1.4⌬ex p45,47, it was possible that our co-immunoprecipitation assay did not report subtle changes in CaBP4 binding affinity. Therefore, we compared CaBP4 binding to these variants in an ELISA binding assay. For these experiments, we generated SUMO-tagged fusion proteins corresponding to the CT of Ca v 1.4 FL and Ca v 1.4⌬ex p45,47 and compared their binding to 96-well plates coated with GST-tagged CaBP4. Binding of Ca v 1.4⌬ex p45,47 CT to CaBP4 was similar to that by Ca v 1.4 FL CT (Fig. 6B)  We next determined if deletion of exon 47 affected modulation by CaBP4. For these experiments, we used Ca v 1.4⌬ex 47 because the splicing of exon 45 had no effect on the electrophysiological properties of the channel (Figs. 4 and 5). Although CaBP4 causes a negative shift in voltage-dependent activation of Ca v 1.4 FL (31, 32), it did not similarly affect Ca v 1.4⌬ ex47. There was no significant difference in I-V or normalized tail current-voltage relationships in cells expressing Ca v 1.4⌬ex 47 alone and those co-expressing CaBP4 (Fig. 7, A and B; Table 4). However, CaBP4 did blunt the strong inactivation of Ca v 1.4⌬ ex47 I Ca . CaBP4 caused a significant 2-fold reduction in the amount of I Ca inactivation of Ca v 1.4⌬ ex47 (Fractional I Ca ϭ 0.30 Ϯ 0.1, n ϭ 5 for Ca v 1.4⌬ ex47 alone versus 0.56 Ϯ 0.10, n ϭ 7 for Ca v 1.4⌬ ex47 ϩ CaBP4; p Ͻ 0.02 by t test ; Fig. 7C). We      (7) Norm.
ICa Norm. Ϫ80 mV to various test voltages with 2-ms repolarization to Ϫ60 mV during which the peak tail current amplitude was measured. Current amplitudes were normalized to that evoked by a ϩ60-mV pulse and plotted against the test voltage. Parentheses indicate the number of cells.  acids (aa) of the channel protein (19,22,43). The underlying mechanism is controversial and involves binding of the CTM to a site in the proximal CT, which may physically displace CaM from the channel (22,43). Alternatively, the CTM binding to the proximal CT is not competitive, but allosterically alters the binding of CaM in a way that weakens CDI (20). This interaction of the CTM with the proximal CT could be affected by sequences between the two domains, including exon 45 (Fig. 1). However, we did not find that splicing out part of exon 45 affected CDI or voltage-dependent activation (Figs. 4 and 5). These results are consistent with previous analyses of the partial deletion of exon 45 in chimeric Ca v 1.2-Ca v 1.4 (23), and with the inconsequential effects of alternative splicing of the analogous exon 44 of Ca v 1.3 (43).

ICa
By contrast, deletion of exon 47 had dramatic effects on CDI. For Ca v 1.4⌬ex 47 and Ca v 1.4⌬ex p45,47 variants (Fig. 4), CDI was as robust as that caused by removal of the entire CTM (19,20,22). At first glance, this result may seem at odds with previous findings that truncation of the final 55 aa distal to exon 47 disabled the ability of the CTM to suppress CDI (22). Because deletion of the last 32 amino acids was ineffective in this regard, it was concluded that the stretch of 20 aa between aa Ϫ55 and Ϫ32 from the C terminus contains the molecular determinants for CDI suppression (19,22). Our results show that these 20 aa are not sufficient to support the function of the CTM because their presence in Ca v 1.4⌬ex47 and Ca v 1.4⌬ex p45,47 was not able to suppress CDI (Fig. 4, C and D). The region encoded by exon 47 may enable proper folding of the CTM and/or provide key contact points required for the intramolecular interaction with the proximal CT. Consistent with both possibilities, deletion of portions of exon 47 prevented binding of the CTM to the proximal CT (20).
In addition to suppressing CDI, the CTM inhibits voltage-dependent activation of Ca v 1 channels. For Ca v 1.2, the distal CT is proteolytically cleaved but remains noncovalently attached to the proximal CT, causing a positive shift in V h (45). Although there is no evidence that the distal CT is cleaved in vivo for Ca v 1.3 (46) or Ca v 1.4, the distal CT of these channels autoinhibits voltage-dependent activation. Ca v 1.3 or Ca v 1.4 mutants or splice variants lacking the CTM exhibit negative shifts in V h compared with full-length channels (19,20,22,23,43,44). Our findings indicate that exon 47 is a key element within the CTM that regulates activation because V h for Ca v 1.4 variants lacking exon 47 were ϳ15 mV more negative than that of Ca v 1.4 FL (Fig.  5, Table 3). How the CTM regulates voltage-dependent activation of Ca v 1.4 is not entirely clear but could involve inhibition of movement of the voltage-sensing domains. However, for Ca v 1.2 and Ca v 1.3, the positive shift in V h due to autoinhibition by the CTM is attributed to weaker coupling of voltage sensor movement to opening of the channel pore (45,47). Addressing the underlying mechanism for Ca v 1.4 would require analysis of the voltage dependence of gating charges representing movement of the voltage sensors (48). These experiments would be technically quite challenging for Ca v 1.4 channels, which produce very modest current density in heterologous expression systems compared with Ca v 1.2 and Ca v 1.3 (40).  (31,32). The inability of CaBP4 to similarly promote voltagedependent activation of channels lacking exon 47 (Fig. 7, A and  B) indicates a key role for this exon in supporting CaBP4 modulation. CaBP4 still binds to the CT (Fig. 6B) and markedly suppresses CDI of channels lacking exon 47 (Fig. 7, C and D), which argues against the possibility that deletion of exon 47 prevents the physical interaction of CaBP4 with the channel. Our results agree with previous findings that deletion of the entire previously defined CTM does not abrogate the physical interaction of CaBP4 with Ca v 1.4 FL , despite preventing effects of CaBP4 on voltage-dependent activation (32). As discussed for its role in regulating CDI, exon 47 may contribute to the structure and/or function of the CTM, which is necessary for   (40). Although other ␤ and ␣ 2 ␦ subunits may be expressed in the retina (50), strong CDI and enhanced voltage-dependent activation are seen upon removal of the CTM from Ca v 1.4 channels coexpressed with ␤ 3 and ␣ 2 ␦ 1 subunits (22). Therefore, these properties in Ca v 1.4⌬ex 47 and Ca v 1.4⌬ex p45,47 are not likely to be significantly affected by differences in auxiliary subunit composition.
To compensate for the relatively small currents carried by Ca v 1.4 compared with other Ca v channels, we used a high concentration (20 mM) of Ca 2ϩ or Ba 2ϩ in the extracellular recording solution. Due to charge screening effects (51,52), this would cause channels to activate at more depolarized voltages than in physiological solutions. Based on activation properties of Ca v 1.4 in 2 mM extracellular Ca 2ϩ (13), the V h values reported here (Tables 2-4) should be ϳ20 mV more positive than would be expected for Ca v 1.4 channels in vivo. Taking this into account, the channels lacking exon 47 would be expected to support ϳ3-fold higher levels of Ca 2ϩ influx compared with Ca v 1.4 FL at the photoreceptor membrane potential in darkness (Ϫ30 to Ϫ40 mV (14,15)). However, this difference may be offset by the presence of CaBP4 in photoreceptor terminals. As a consequence of modulation by CaBP4 (31, 32), Ca v 1.4 FL should exhibit hyperpolarized voltage dependence of activation similar to that of Ca v 1.4⌬ex 47 and Ca v 1.4⌬ex p45,47 (Fig. 5, E  and F). The negative activation properties of Ca v 1.4 FL modulated by CaBP4, and exon 47-lacking Ca v 1.4 variants would promote presynaptic Ca 2ϩ influx to support glutamate release at the photoreceptor membrane potential in darkness. This in turn would ensure mGluR6-mediated closure of nearly all postsynaptic TRPM1 channels, which at the rod-rod bipolar cell synapse, is necessary for the optimal encoding of dim light signals (53,54). At the same time, CaBP4 would suppress CDI of Ca v 1.4⌬ex 47 and Ca v 1.4⌬ex p45,47, much like the CTM does for Ca v 1.4 FL (Fig. 4). Thus, both exon 47-lacking channels and Ca v 1.4 FL could mediate sustained Ca 2ϩ influx necessary for tonic glutamate release in darkness, although via different mechanisms.
We do not discount the possibility that splicing of exons 45 and 47 could have effects in photoreceptors that are independent of the electrophysiological findings in our study. Like other ion channels, Ca v channels interact with a variety of modulatory and scaffolding proteins that collectively regulate the cellular roles of these channels in different tissues (55). Because the CT of Ca v 1 channels is a major hotspot for such protein interactions, deletion of exon 47 and/or partial deletion of exon 45 could disrupt the association of the channel with a regulatory protein present in photoreceptor terminals that is not endogenously expressed in our heterologous expression system (i.e.

Experimental Procedures
Plasmids and Cloning of Human Ca v 1.4 Variants-The cloning of the human full-length Ca v 1.4 ␣ 1 subunit (Ca v 1.4 FL ) with an N-terminal FLAG epitope (GenBank TM number AF201304) in pcDNA3.1 vector, ␤ 2ϫ13 (GenBank TM number AF465485), ␣ 2 ␦ 4 (GenBank TM number NM_172364), and CaBP4 (Gen-Bank TM number AY 039217.1) was described previously (31,40). For the cloning of Ca v 1.4 deleted from part of exon 45 (ex p45) and full exon 47 (ex 47; Ca v 1.4⌬ex p45,47), a similar strategy to that used for the Ca v 1.4 FL was followed (40 Technologies). To remove the CaM binding site(s), a fragment encoding amino acids 1603 to the stop codon was also amplified. These PCR products were cloned into the pET-SUMO vector (Life Technologies) for fusion to both a His 6 and a SUMO tag. The fusion proteins were expressed in BL21(DE3)pLysS Escherichia coli after induction with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside and purified on nickelnitrilotriacetic acid columns according to the manufacturer's protocol.
CaBP4 was amplified by PCR from a human retina cDNA library and cloned into pentr-D-TOPO vector (Life Technologies). After sequencing, the cDNA was transferred by recombination into the pDest15 vector using the Gateway Technology System (Life Technologies) for fusion to a GST tag and expression in bacteria. The GST fusion proteins were expressed in BL21(DE3) pLysS E. coli after induction with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside and purified on a glutathione column according to the manufacturer's protocol.
Quantitative PCR Analysis of Human and Monkey Ca v 1.4 and Their Splice Variants-All procedures were approved by the Institutional Animal Care and Use Committee of the University of Washington. Retinas from Macaca nemestrina were obtained at the University of Washington Regional Primate Center (Seattle, WA). Human retinas were obtained from donors without known eye disease from the Lions Eye Bank of Oregon 4 -10 h after death. Total RNA was isolated from the retina of human, monkey, or mouse using a RNeasy kit (Qiagen). The relative expression of splice variants was determined by a two-step quantitative PCR. Total RNA (1 g) was subjected to first strand cDNA synthesis using SuperScript III reverse transcriptase and oligo(dT) in a volume of 20 l according to the manufacturer's protocol (Life Technologies). For the qPCR analysis of the Ca v 1.4⌬ex p45,47, primers were designed on the exon 44-alternate exon 45 joint (forward) and ϳ200 bp downstream on the exon 46-exon 48 joint (reverse,  (Table 1, rows 2 and 7-9). For normalization, primers were used to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Table 1, rows 10 to 12). Reactions were carried out in triplicate using 0.5 l of cDNA, 400 nM of each primer, and 10 l of QuantiTect SYBR Green PCR mix (Qiagen) in a 20-l total reaction volume. After an initial incubation at 95°C for 15 min, the qPCR was carried out for 40 cycles of denaturation at 95°C for 15 s, annealing at 68°C for 30 s, and extension at 72°C for 1 min on a ABI PRISM 7000 (Applied Biosystems). Single bands of the predicted size were verified by agarose gel electrophoresis. Threshold cycle was determined using the ABI Prism 7000 software. Data were analyzed by comparing cycle threshold (C t ) normalized to the C t values of the internal control, GAPDH (⌬C t value ϭ C t value of WT or variant Ϫ C t value of GAPDH); standard deviation of ⌬C t ϭ ͌(S.D. of variant or WT) 2 ϩ (S.D. of GAPDH) 2 (41).
Co-immunoprecipitation of Ca v 1.4 and CaBP4 -HEK-293 cells were transfected with cDNAs encoding Ca v 1.4 FL or Ca v 1.4⌬ex p45,47, ␤ 2ϫ13 , ␣ 2 ␦ 4 , and CaBP4. Three days later, whole cell lysates were prepared by incubation of transfected cells at 4°C for 1 h in 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM MgCl 2 , 0.1 mM CaCl 2 and inhibitors of proteases (Sigma). Lysates were subject to centrifugation at 22,000 ϫ g for 30 min and incubated with mouse IgG (purified on protein G plus agarose from mouse serum) or anti-FLAG antibodies (Sigma). After 1 h incubation at 4°C, protein G-magnetic beads (Life Technologies) were added and the incubation proceeded for 3 h at 4°C. After 4 washes with lysis buffer, proteins were eluted with SDS-sample buffer and analyzed by Western blotting with specific antibodies.
Enzyme-linked Immunoadsorbent Assay (ELISA)-Purified GST, GST-CaBP4, or GST-CaM fusion proteins (2 g/ml in 100 mM sodium bicarbonate, pH 9.0) were bound to 96-well ELISA plates (200 ng/well) overnight at 4°C. The wells were blocked with animal-free blocker (Vector laboratories) for 1 h at room temperature. 2-Fold dilutions of SUMO-Ca v 1.4 C-terminal domain (CT) fusion proteins in TBST_MC (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.05% Tween 20) were added and reactions were incubated for 1 h at room temperature. After 3 washes in TBST-MC, bound Ca v 1.4 CT was detected with rat anti-SUMO antibodies (raised in rats and purified with SUMO proteins using a previously described method (40)) for 1 h at room temperature, followed by incubation with alkaline phosphatase-conjugated anti-rat antibodies. Reactions were incubated with p-nitrophenyl phosphate substrate (diluted in 100 mM Tris, pH 9.0, 50 mM MgCl 2 , 100 mM NaCl) for 30 min at room temperature and the absorbance was measured at 405 nm with a microplate reader (Bio-Rad). The absorbance data of nonspecific binding of Ca v 1.4 to GST (negative control for binding to GST) was subtracted from that for binding of SUMO-Ca v 1.4 CT to GST-CaBP4.
We have found that overexpression of CaBP4 has inhibitory effects on Ca v channel current density, perhaps through dampening of channel expression levels. To offset these effects, we used an ecdysone-inducible system to co-express CaBP4 with Ca v 1.4 (27). Cells were co-transfected with Ca v 1.4 subunits as described above, but cotransfected with CaBP4 subcloned into an ecdysone-inducible expression (pIND) vector (Invitrogen; 3 g) and pVgRXR (1 g), which encodes a heterodimeric retinoid X receptor (RXR) and ecdysone receptor (VgEcR). After 24 h, transfected cells were treated with an ecdysone analog, Ponasterone A (10 M; Thermo-Fisher Scientific) or 1% ethanol (control) for 8 -10 h to induce CaBP4 expression.
Whole cell patch clamp recordings were performed at room temperature between 48 and 72 h after transfection. Data were obtained under voltage-clamp with an EPC-9 patch clamp amplifier operated by either Patchmaster or PULSE software (HEKA Elektronik) and analyzed with Igor Pro software (Wavemetrics). External recording solutions consisted of (in mM): Tris (140), CaCl 2 or BaCl 2 (20), and MgCl 2 (1). Internal pipette solution consisted of (in mM): N-methyl-D-glucamine (140), HEPES (10), MgCl 2 (2), Mg-ATP (2), and EGTA (5). The pH of external and internal recording solutions was adjusted to 7.3 with methanesulfonic acid. Pipette resistances were typically 2-4 megohms, and series resistance was compensated up to 70%. Leak subtraction was conducted using a P/4 protocol. Statistical analysis (Student's t test, Mann-Whitney rank sum test, or by a one-way ANOVA) was done and graphs were made with SigmaPlot (Systat Software). All averaged data represent mean Ϯ S.E., and result from at least 5 independent transfections.
Author Contributions-F. H. and B. W. performed experiments and analyzed data. A. L., F. H., and B. W. contributed to experimental design and writing and approval of the final manuscript.