JBC Papers in Press. Published on July 31, 2012 as Manuscript M112.362509 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.362509
A short polybasic segment between the two conserved domains of the β 2a-subunit modulates the rate of inactivation of R-type calcium channel*S Erick Miranda-Laferte‡, Silke Schmidt‡, Antonella C Jara §, Alan Neely§1, Patricia Hidalgo‡1 From the ‡Institut für Neurophysiologie, Medizinische Hochschule Hannover, Hannover, Germany and the §Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso; Valparaíso, Chile Running title: New determinant for modulation of inactivation in the β2a-subunit 1
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Keywords: channel; voltage-dependent inactivation; beta-subunit; Protein palmitoylation
SUMMARY Besides opening and closing, high-voltage activated (HVA) calcium channels transit to a non-conducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of deleterious rise in calcium concentration. R-type HVA channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular β-subunit isoform, β 2a, inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N-terminal anchors β2a to the plasma membrane. The current
view is that membrane-anchored β 2a immobilizes the channel inactivation machinery and confers slow-inactivation phenotype. β-subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here we indentified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient β 2a to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slowinactivation conferring phenotype. The linker upstream the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the β 2a-subunit that might involve electrostatic interactions with an unknown target on the channel´s inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of β 2a-subunit.
1 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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Background: Membrane-anchoring underlies inhibition of voltage-dependent inactivation (VDI) of calcium channels by β2a-subunit. Results: A polybasic segment of β2a slows VDI without membrane association. This effect is abolished by charge neutralization. Conclusion: VDI inhibition by β 2a-subunit can occur without membrane-anchoring by a mechanism that relies on positively charged residues. Significance: A novel mechanism underlying inhibition of VDI in calcium channels is revealed.
Crystallographic studies from three of the four known CaVβ isoforms (CaVβ 1 to CaVβ4) revealed the molecular aspects of this interaction. CaVβ shares a common structural arrangement with members of the Membrane Associated Guanylate Kinase family (MAGUK): a highly conserved Src homology 3 (SH3) and guanylate kinase (GK) domain flanked and joined by variable segments (13-15). The binding motif in the pore-forming subunit, AID, forms a α-helix that penetrates into a hydrophobic groove within the GK domain. In contrast to the structural core of the protein, namely SH3-GK unit, the structural data for the variable regions and their interactions with the rest of the protein are, as yet, unknown. Functionally, all β-subunits shift the voltage-dependence of
activation toward more negative voltages but they differentially modulate inactivation of HVA CaVα1 sub-types (6,8). The native R-type CaV2.3 channel, originally cloned from human and mouse brain (16), inactivates very fast upon depolarization and, in heterologous expression systems, most CaVβs speed up inactivation even further. CaVβ2a, hereby referred as to β2a, is the only isoform that slows inactivation and is also the only isoform that undergoes palmitoylation (6,17). This splice variant encompasses two adjacent cysteine residues at positions 3 and 4 within its N-terminal variable region. These are dynamically palmitoylated and target the protein to the plasma membrane (17-20). In oocytes expressing CaV2.3 channels, β2a induces a 13-fold increase in the time for the current to return to 50% of its peak amplitude evoked by depolarizing pulse to 0 mV (hereby referred as to T0.5). Even after 15 seconds, inactivation is still incomplete; leaving around 10% of residual current (6). Substituting the two cysteines by alanines impairs membrane localization and also abolishes this slow inactivation phenotype (17,18). The prevalent view is that anchoring of β2a to the membrane slows down channel inactivation by immobilizing the inactivation machinery and, hence, preventing it from occluding the conduction pathway (19,21). However, it was shown later that the slow inactivation-conferring phenotype can be manifested in non-membrane-anchored CaVβ, suggesting that this is not the only mechanism that contributes to slowing down VDI (7,22,23). The same studies also implicate the linker joining the SH3 and GK domains in VDI regulation. Among the possible mechanisms that come to mind is that the linker contributes to anchoring the β-subunit to the plasma membrane, so that it interacts directly with the inactivation machinery or modifies the interaction of SH3-GK core with the channel protein. Here, we used Xenopus oocytes to study the effect of different CaVβ-derivates on VDI and tsA-201 cells in order to investigate the intracellular localization of these protein constructs. The rationale is that while mammalian cells are well suited for confocal microscopy they are too small for protein injection. The opposite is true for Xenopus oocytes. Moreover, expression of CaVα1 in
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The entry of calcium ions into the cell triggers a multitude of cellular responses that rely on a tight spatio-temporal regulation of the calcium transient spreading within the cell for their coordination (1,2). High-voltage activated (HVA) calcium channels open in response to large membrane depolarization and constitute the major entry pathway for calcium into excitable cells. The largest component of HVA channels, α1 subunit (CaVα1), contains the ion conduction pore, the voltage-sensor, and multiple intracellular domains that regulate calcium influx and provide interaction sites for regulatory proteins. Two subfamilies of HVA CaVα1 are recognized: CaV1.x or L-type channels and CaV2.x, also referred as to neuronal channels (CaV2.1 encoding P/Q-type; CaV2.2 encoding N-type; and CaV2.3 encoding Rtype) (3). Following strong depolarization and calcium permeation, HVA channels enter into an inactivated non-conducting state which constraints the amount of calcium influx and protects the cells from the cytotoxic effects of an excessive calcium rise. Inactivation of HVA channels is triggered by both, calcium increase and prolonged membrane depolarization, referred as to calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI), respectively (4). While CDI depends on the binding of calmodulin to a conserved site among HVA channels (5), VDI is an intrinsic property of CaVα1, but is strongly modulated by the regulatory β-subunit (CaVβ)(611) which binds to a site highly conserved among HVA channels, termed the α-interaction domain (AID)(12).
mammalian cells is sensitive to the co-expression of the β-subunit. In contrast, in Xenopus oocytes, association with the CaVβ does not seem to be a requirement for the pore-forming subunit to reach the plasma membrane. In fact, CaV2.3 bearing a mutation in the primary β-subunit anchoring site expresses as well as wild-type channels (24). This is also true for CaV1.2 channels (25,26,27). Using this approach, we identified within the linker region of the β-subunit a positively charged segment, twelve amino acids long, that slows down inactivation without conferring membraneanchoring to the protein.
EXPERIMENTAL PROCEDURES Construction of cDNA and protein expressioncDNA encoding all CaVβ2a (Swiss-Prot: Q8VGC32, described elsewhere (27)) and CaVβ3xo (SwissProt: Q91630) derivatives were subcloned by PCR methods into pRSET vector (Invitrogen) to include N-terminal Histidine tag and Xpress-epitope tag. The proteins were over-expressed in bacteria and purified by metal-affinity chromatography, followed by size-exclusion chromatography onto a Superdex S-200 column (GE Healthcare Life Sciences), as described (27). Proteins were concentrated to 0.7-5.0 mg/ml by ultra-filtration, fast frozen and stored at -80°C until use. For confocal fluorescence microscopy the same cDNAs encoding for the different CaVβ2a derivatives used for protein expression were subcloned into pcDNA 3.1 vector and fused to YFP. All constructs were verified by DNA sequencing. The human form of CaV2.3 subunit (Swiss-Prot entry Q15878) used in this study has been previously described (28,29).
Oocytes injection and electrophysiological recordings- The cRNA encoding for CaV2.3 α1 subunit was synthesized using a capping RNA transcription kit (mMESSAGE mMACHINE™, Ambion) and injected into Xenopus laevis oocytes, as previously reported (30). To minimize cRNA degradation the protein was injected 1 hour after cRNA injection, using the same method as for cRNA injection. Electrophysiological recordings
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The effect of this linker segment was abolished by neutralizing the charged residues and masked by the upstream linker region, but not by the other variable (N- and C-terminus) segments of the protein. We propose the existence of electrostatic interactions between the polybasic linker segment and the channel inactivation machinery itself, or its regulatory components that contribute to the inhibition of VDI by β2a subunit. The exposition of this segment appears to be regulated by the palmitoylated state of the N-terminus of β2a.
Western Blot analysis- For Western Blot analysis of β2a-derivative protein constructs five non-injected control oocytes and five oocytes injected with purified His-β 2a protein constructs five days before recording. Oocytes were transferred to a 1.5 ml tube and homogenized by pipetting up and down with 50 µl lysis buffer (100 mM sodium phosphate, pH 7.2), supplemented with 0.1% Triton X-100 and protease inhibitor cocktail (Sigma). After incubation on ice for 15 minutes (15 seconds vortexing every 5 minutes) the samples were centrifuged twice at 12,000 rpm in a microcentrifuge for 10 minutes at 4°C. The supernatant was diluted with 5 X SDS loading buffer and loaded on a 12% acrylamide gel. After SDS-PAGE electrophoresis, the proteins were electrically transferred to ECL Plex membranes (GE Healthcare Life Sciences) for one hour. Membrane was blocked using 3% BSA in TBS buffer (10 mM Tris, 150 mM NaCl, pH 7.5) and incubated with primary antibody diluted according to the manufacturer´s instructions in TBS supplemented with 1% BSA. Three primary antibodies were used depending on the β-construct to be detected; anti-calcium channel β 2 subunit antibody (Sigma) when the C-terminus was present (β2a C3,4S, β2a SH3-PBLK-GK + CT, β2a ΔPBLK), anti-CACNB2 antibody (Abcam) only when the full linker sequence was present (β2a SH3-LK-GK) and anti-Xpress antibody (Invitrogen) for the rest of the constructs (β2a SH3PBLK-GK, β 2a SH3-PBLK-GK + ΝΤC3,4S, β2a SH3PBLKAla-GK, β2a SH3-GK). Immuno-detection was carried out using HRP-conjugated secondary antibody (Pierce Antibodies, Thermo Scientific) diluted 1:40.000 to 1:60.000 and a chemiluminescent detection kit (SuperSignal West Femto Chemiluminescent Substrate, Thermo Scientific). Membranes were exposed between 1560 seconds into a GeneGnome chemiluminescence imaging system (SYNGENE).
were performed using the cut-open oocyte technique (31) with a CA-1B amplifier (Dagan Corp., Minneapolis MN USA), as described (27). Data acquisition and analysis were performed using the pCLAMP system and software (Axon Instruments Inc., Foster City CA USA). Linear components were eliminated by P/-4 pre-pulse protocol. All experiments were carried out in at least two batches of Xenopus laevis oocytes from two different frogs. The external solution contained in mM, 10 Ba2+, 96 n-Methylglucamine, and 10 HEPES and the internal solution 120 nMethylglucamine, 10 EGTA, and 10 HEPES. Both solutions were adjusted to pH 7.0 with methanesulfonic acid.
RESULTS Co-injection of full length β-protein with α1cRNA fully reconstitutes β-subunit modulation of activation and inactivation of CaV2.3 R-type calcium channel expressed in Xenopus oocytesInstead of injecting the cRNA encoding for CaVα1 and CaVβ, that is, the widespread strategy to study β-modulation of calcium channels, we injected βderived constructs as purified proteins together with the cRNA encoding the α1 subunit (27,29,30). With this strategy we were able to study isolated domains and short constructs of β2a that are not functional when injected as cRNA (29), but that regulate calcium channel function when injected as purified proteins. To confirm the integrity and folding of the recombinant proteins, all constructs used in this study were subjected to SDS-reducing PAGE and size-exclusion chromatography (Supplemental Fig. S3).
As observed from earlier co-injection experiments of cRNA encoding α1 and β-subunits, β2a inhibits VDI of CaV2.3 channels expressed in Xenopus oocytes while β3xo stimulates it (8,33-35). This effect is reflected in much greater values of T0.5 for CaV2.3/β2a channel complexes than CaV2.3/β3xo and in steady-state inactivation curves, exhibiting opposing voltage shifts with respect to the CaV2.3 α1-subunit alone (Fig. 1B, C and Table 1). Besides, shifting the steady-state inactivation curve to more depolarizing potentials β2a also induces a residual non-inactivating current component that is unique to this isoform (Fig. 1C). In contrast to their differences in the channel inactivation curve, β2a and β3xo shift the activation curve to the same extent and direction (Fig. 1D and Supplemental Table S1). This change in the midpoint of the activation curve towards more hyperpolarizing potentials is the signature of βsubunit modulation of HVA calcium channels (33,36) and we used it as a parameter to confirm the functional state of the protein construct within the oocytes. We conclude that the effect of βsubunit protein on CaV2.3 channels is undistinguishable from the one of cRNA source when injected with CaVα1cRNA. This, however, is not the case for β-subunit protein injected at a later stage of CaVα1 biogenesis (29). The distal linker segment of CaVβ2a comprising the amino acid sequence HSKEKRMPFFKK slows down VDI of CaV2.3 channels- Previous results from Qin et al. and Richards et al., (7,22) suggested that the linker joining SH3 and GK domains of the β-subunit plays a role in the regulation of VDI. However the variability in length and sequence of this region makes it difficult to define further the structural determinant of VDI inhibition. Eye inspection of
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Confocal fluorescence microscopy- Live cell confocal imaging was carried out with a 40x oil immersion objective on a Leica inverted confocal microscope equipped with an argon-ion laser using transiently transfected tsA201 cells. Cells were cultivated on a glass coverslip and imaged 18 to 48 hours after transfection using Lipofectamine™ (Invitrogen) according to the manufacturer`s instructions. For excitation of YFP the 514-nm laser was used and the emitted light was monitored between 520-560 nm. β2a –YFP has been described elsewhere (32).
Figure 1 illustrates the strategy followed in this study to investigate the effect of different βsubunit derivatives on VDI of R-type CaV2.3 channels. Protein constructs were injected into Xenopus oocytes one hour later than the cRNA encoding the α1 subunit, so as to avoid RNA degradation. Electrophysiological recordings were performed five to six days after cRNA/protein injection, corresponding to the time necessary for the α1-subunit to be assembled in the plasma membrane (Fig. 1A).
To test if indeed the polybasic segment is responsible for this conferring-phenotype we generated a linker-less β-subunit construct, consisting of only SH3 and GK domains (β2a SH3GK, Fig. 2B) that has been proposed earlier to be the functional unit of the β-subunit (13,37). In contrast to β2a SH3-PBLK-GK, β2a SH3-GK confers a fast inactivating phenotype to CaV2.3 channel complexes, even faster than the channel alone (Fig. 2C and Table 1). As performed for the full-length proteins (see Fig. 1D), the functional state of the β-derivatives constructs five days following injection into oocytes was tested by measuring the activation curves of the different CaV2.3 channel complexes. Both, β 2a SH3-PBLKGK and β2a SH3-GK produced a left-shift in the activation curve of around 10 mV accompanied with an increase in the slope, and relative contribution of the first component of the sum of two Boltzmann distributions that fit the activation curves (Supplemental Fig. S1 and Table S1). We also verified the integrity of the β-derivatives by Western Blot analysis (Fig. 2B, inset). While no signal was detected in lysates from non-injected oocytes, a strong band that migrated at the same position as the purified protein was visible in lysates from injected Xenopus oocytes showing that β-derivatives constructs were stable for at least five days within the oocyte (Fig. 2B, inset). Although only β2a SH3-PBLK-GK slowed inactivation, both constructs shifted the voltage-
dependence of VDI towards more negative voltages, compared to CaV2.3 expressed alone. The voltage for 50% inactivation (V1/2) was shifted by about 8 mV to the left by the presence of β2a SH3-PBLK-GK and 20 mV by β2a SH3-GK (Fig. 2D and Table 1). Nevertheless, as with wild type β2a, a residual current can still be detected at the end of a 10 s pulse in CaV2.3/β2a SH3-PBLK-GK expressing oocytes. These results identify the distal polybasic linker segment of CaVβ2a as a structural determinant for slowing down inactivation of CaV2.3 channels and reveal divergences in the modulation of kinetic and steady-state parameters. The polybasic linker segment that slows down inactivation does not confer membrane-anchoring. The interaction of membrane-associated proteins with lipid membranes can be driven by electrostatic interactions between positively charged residues and negatively charged lipid membranes (38). The basic nature of PBLK might provide attachment of the β-subunit to the plasma membrane, mimicking the mechanism proposed for palmitoyl-membrane-anchored β2a (17). To investigate the cellular location of β2a SH3-PBLKGK we fused it to YFP (β2a SH3-PBLK-GK-YFP), expressed it in mammalian cells and visualized it by confocal microscopy (Fig. 3). In clear contrast to wild-type β2a-YFP that is located at the plasma membrane, fluorescently labeled β2a SH3-PBLKGK is distributed throughout the cytoplasm, closely resembling the distribution of nonpalmitoylable β2a C3,4S double mutant (Fig. 3). To rule out that the His-tag used to purify the recombinant proteins may interfere with palmitoylation of β2a we fused the His-tagged β2a sequence to YFP and expressed it in tsA201 cells. Confocal images of tsA201 cells expressing Hisβ2a-YFP show that this protein is indeed targeted to the plasma membrane (Supplemental Fig. S4). Thus, the polybasic linker confers slow inactivation phenotype by other means, not only providing a membrane-anchoring domain to the protein. The positively charged amino acids within the polybasic linker segment are required for slowing down inactivation of CaV2.3 channels- Since the most striking property of this segment is a relatively high density of positively charged
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the amino acid sequence of the linker region of β2a reveals a short polybasic segment at the boundary of the GK domain, comprising residues 202 to 213, hereby referred to as polybasic linker segment (PBLK, Fig. 2A) that is absent in β3xo. To isolate the potential impact of the polybasic linker segment on VDI regulation, we removed all other variable regions of β2a, including the N- and C-termini and the segment upstream PBLK that encompasses a long serine-rich sequence (PSLK, Fig. 2A), containing several predicted phosphorylation sites. The result is a construct that consists of SH3 and GK domains joined only by PBLK (β2a SH3-PBLK-GK, Fig. 2B). We found that this construct confers slow-inactivation phenotype, despite lacking the palmitoylable NT segment that is commonly recognized as responsible for this effect.
residues we rationalized that they might still be functionally relevant although not for surface membrane association. To test this idea we substituted all positively charged residues within this segment by alanine (β2a SH3-PBLKAla-GK, Fig. 4A). This β-derivative remains intact after five days of injection into Xenopus oocytes, as judged by Western Blot analysis (Fig. 4A) and left-shifts of the channel activation curve (Supplemental Fig. S1 and Table S1).
The linker region upstream the polybasic linker segment masks its effect on VDI- It remains to be explained why non-palmitoylated β2a C3,4S double mutant (β2a-C3,4S) containing the polybasic linker segment confers fast inactivation to CaV2.3 channels (17,29) (Fig. 5A, E). This observation implies that other parts of the nonmembrane-anchored β-proteins counteract the effect of the polybasic linker segment. We followed a simple strategy to identify variable segments of the protein that may occlude the effect of the polybasic linker segment. This involved adding to β2a SH3-PBLK-GK the rest of the variable regions, one at a time (Fig. 5 and see also Fig.2A). Three new constructs were designed; β2a SH3-PBLK-GK plus NT variable region carrying the double cysteine substitution (β2a SH3-PBLKGK + ΝΤC3,4S), β2a SH3-PBLK-GK plus CT variable region (β2a SH3-PBLK-GK + CT) and β2a SH3-PBLK-GK lacking NT and CT but carrying the full linker (β2a SH3-LK-GK). Adding NT or CT to β2a SH3-PBLK-GK did not interfere with the ability of PBLK segment to confer slow-inactivation phenotype, and in the presence of either constructs the channel voltagedependence of inactivation is closer to the parent protein than to β2a C3,4S (Fig. 5A-C, E-F and Table 1). In contrast, β-subunit carrying a fulllength linker while lacking the N and C termini,
The idea that a posttranslational modification may be involved in occluding a slow-inactivation phenotype is appealing because it might explain why non-membrane-anchored β-subunit can slow inactivation when acutely applied to already expressed CaV2.3 channels (29). Since the linker region upstream the polybasic segment contains several predicted phosphorylation sites, there is a possibility that the addition of phosphate groups would hinder potential electrostatic interactions which might, in turn, be responsible for VDI inhibition by the polybasic segment. We substituted all serine and threonine residues by alanine within PSLK in β2a-C3, 4S background, and no effect on T0.5 or the inactivation curve was observed (Supplemental Fig. S2 and Table S1). This indicates that phosphorylation events within the region upstream the PBLK do not participate in VDI modulation. Although the molecular mechanism by which the upstream linker region hinders the effect of the distal polybasic segment remains elusive, the above results provide a clear explanation why nonmembrane-anchored full-length proteins containing the polybasic segment do not manifest their phenotype. Palmitoylated β2a lacking the polybasic linker segment inhibits voltage-dependent inactivation of CaV2.3 channel complex to a lesser extent than does the wild-type protein. It is well accepted that membrane-anchoring by itself suffices to confer slow inactivation to CaV2.3 subunit. This idea is based on results showing that removal of the Nterminal double cysteine motif in β2a abolishes membrane localization and slow-inactivation conferring phenotype which is rescued by artificially anchoring β2a C3,4S to the membrane (17,19) (see also Figs. 3 and 5). Within this framework, the functional relevance of PBLK on membrane-anchored β2a-subunit comes into question. In order to provide an answer to this we removed the PBLK from wild-type β2a containing the double cysteine motif (β2a ΔPBLK). This
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Channel complexes bearing β2a SH3PBLKAla-GK inactivates nearly as fast as the construct lacking this segment (Fig. 4B, C and Table 1). These results demonstrate that the basic residues within the PBLK segment are required for conferring slow-inactivation phenotype to nonmembrane-anchored β-subunit and suggest the involvement of electrostatic interactions in VDI modulation.
gives rise to relatively fast inactivating CaV2.3 channels and the inactivation curve is shifted even further to the left than with β2a C3,4S, indicating that the sequence upstream the polybasic segment occludes its effect on VDI (Fig. 5D-F and Table 1).
protein was also stable within the oocyte, as judged by Western Blot analysis and its effect on the channel’s voltage-dependence of activation (Fig. 6A and Supplemental Table S1). CaV2.3/β2a ΔPBLK channels inactivated slightly faster than CaV2.3/β2a channel complex and when fused to YFP, β2a ΔPBLK still localized at the plasma membrane (Fig. 6A-C). Thus, dissociation of the membrane does not explain this effect. Although the reduction in T0.5 is relatively modest it is also accompanied by a left-shift in the voltage to reach 50% inactivation and a reduction in the noninactivating current (Fig. 6D and Table 1). This result indicates that in membrane-anchored βsubunit, the polybasic linker segment contributes to the inhibition of VDI.
The physiological relevance of the precise regulation of calcium influx appears reflected in the redundancy of elements that regulate inactivation of voltage-gated calcium channels. This added complexity barred a full mechanistic account of voltage-dependent inactivation in CaV2.x channels. A simple ball-and-chain model in which the membrane-anchoring of the auxiliary subunit hinders the movement of the ball is rather simplistic and cannot explain why binding of a large non-membrane-anchored subunit may facilitate the process. Although our present study does not provide a definitive answer to this question it does, however, contribute to a better understanding of the modulation of inactivation by the regulatory β-subunit. Four conclusions can be drawn from the present work; i) there is an additional structural determinant in β2a isoform, the distal polybasic linker segment, besides the N-terminal palmitoylable region, that contributes to slowing down inactivation, ii) the polybasic segment inhibits inactivation in a membrane-anchoring independent manner and the basic residues within are required for this effect, iii) the linker sequence upstream the polybasic segment masks the contribution of the latter and iv) the effect of palmitoylated N-terminus of the β-subunit predominates over the PBLK-mediated inhibition, but both act synergistically to inhibit VDI.
We envisage that membrane-anchoring not only contributes to immobilize the inactivation particle, as commonly accepted, but also exposes the polybasic distal linker segment that adds an additional component to VDI inhibition through an electrostatic repulsion of a putative inactivation particle (Fig. 7). In the absence of palmitoylation, as the protein is not longer anchored to the plasma membrane, the linker may fold over, occluding the positive charges of the PBLK and masking their functional association with a potential target within CaVα1. Membrane-anchoring would give rise to an extended conformation of the linker that exposes the positively charged residues of the PBLK (Fig.7). Another explanation is that the polybasic distal linker segment has no direct action on the inactivation machinery, but confers a permissive orientation to SH3/GK functional unit. This idea appears unlikely though in light of our data, showing that several β-constructs bearing no linker or linker segments that differ in length and
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DISCUSSION
Work by others has also shown altering the length and sequence of the linker impact VDI (7,22,23). Qin and collaborators (7) were the first to identify the linker region as a secondary determinant of VDI. Richards and coworkers (22) reported that in the palmitoylation-deficient mutant, removal of the linker speeds inactivation which is expected for a linker encompassing a slow-inactivation conferring sequence, such as PBLK. Stotz and collaborators (23) transferred the linker and GK domain of β3 into β4 and found that VDI of CaV2.2 N-type calcium channel is accelerated. They also found that the N-terminal of the β3 subunit confers fast inactivation to CaV2.2 channels and that transferring this region to β4 is sufficient to accelerate VDI. Interestingly, this 14amino acid region contains five negatively charged residues, however, no positively charged ones. Here we propose that an electrostatic interaction between the polybasic linker and a putative acidic region within either the channel´s inactivation machinery or some of its regulatory determinants slows VDI. An interesting concept is that acceleration of inactivation might involve also electrostatic interactions but of opposite signs, involving a positively charged receptor site in the pore-forming subunit.
sequence composition (β3xo, β2a SH3–GK, β2a SH3-LK-GK, β2a SH3-PBLKAla-GK) confer fast inactivation phenotype. Moreover, crystallographic studies did not reveal any structural differences between the β-subunit alone and complexes to the peptide corresponding to the binding site in the α1-subunit. Unless the relative orientation of SH3/GK domain is different in vivo, one should conclude that there are no major conformational changes associated with the binding of the β-subunit to the channel and that relative orientation of SH3 and GK is irrelevant to inactivation.
An unexpected observation from this study is that constructs capable of inducing a large increase in T0.5, such as β2a SH3-PBLK-GK, produced a modest change in V1/2. Thus, slowing the kinetics of inactivation is not always accompanied by a shift in the voltage-dependence of inactivation toward positive voltages, as one would expect if VDI inhibition occurs by disfavoring inactivated states. Moreover, β2a SH3-PBLK-GK that confers a rather slow inactivation phenotype shifts the steady-state inactivation curve to the left when compared to CaV2.3 alone, as if the equilibrium for the occupation of inactivated states would be favored by the presence of this construct. This suggests that β2a subunit and its derivatives influence not only the stability of the inactivated state but also the energy barriers that the channel must overcome in its way to inactivation. All the available data show that shifting the inactivation curve towards more depolarizing potentials and provoking non-inactivating components at positive voltages require a membrane-anchored β-subunit. Consistent with this view, our studies also show that only palmitoylable β2a displays this behavior. In contrast, the ability to modulate T0.5 does not
In a previous work we showed that acute exposure of CaV2.3 channels to the GK domain of β2a alone was sufficient to slow down inactivation when applied to α1-subunit already assembled into the plasma membrane (29). Unfortunately, the GK domain turned out to be unstable and we could not test whether a long term exposure of this domain, i.e. the cRNA/protein co-injection strategy, would be sufficient to confer fast inactivation. We reported that β-subunits accelerating VDI acquired this phenotype only when co-injecting the protein with α1cRNA. In light of our present results that short constructs also accelerate VDI when co-injected, we rationalized that this could be the case for GK. In favor of this possibility is that the SH3 domain does not have an impact on channel function but on surface expression (25,29). In this scenario, GK would become competent to accelerate inactivation following a determined posttranslational modification, yet to be established. In summary, we were able to identify a new determinant for the inhibition of inactivation within the linker joining the GK and SH3 domains present in all β2 subunits and conserved across different species. This linker has already been pointed out as a potential regulator for inactivation (7,22,23). Here we defined a short segment located in this region that includes positively charged residues. These basic amino acids are required for modulation of VDI through a mechanism that remains to be elucidated and that may involve electrostatic interactions. The functional capability of this segment in non-membrane-anchored β2 subunits remains elusive. One possibility is that the accessibility of PBLK positively charged residues may indeed come about through posttranslational modifications of the protein.
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There is, to date, no direct structural data on the variable regions of β-subunit. Here, we show that N and C- terminal variable segments do not interfere with the effect of PBLK on inactivation and we conclude that such regions do not physically interact with the linker.
require palmitoylation, leading us to conclude that while membrane-anchoring destabilizes the channel inactive state, the linker is geared at regulating the movement of the inactivation gate. This suggests that while the channel transits to the inactivate conformation PBLK comes transiently into close contact with the inactivation machinery.
References
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1. Clapham, D. E. (2007). Calcium signaling. Cell 131, 1047-1058. 2. Carafoli, E. (2002). Calcium signaling: a tale for all seasons. Proc. Natl. Acad. Sci. U. S A 99, 1115-1122. 3. Catterall, W. A. (2000). Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16, 521-555. 4. Cens, T., Rousset, M., Leyris, J. P., Fesquet, P., and Charnet, P. (2006). Voltage- and calcium-dependent inactivation in high voltage-gated Ca2+ channels. Prog. Biophys. Mol. Biol. 90, 104-117. 5. Liang, H., DeMaria, C. D., Erickson, M. G., Mori, M. X., Alseikhan, B. A., and Yue, D. T. (2003). Unified Mechanisms of Ca2+ Regulation across the Ca2+ Channel Family. Neuron 39, 951-960. 6. Olcese, R., Qin, N., Schneider, T., Neely, A., Wei, X., Stefani, E., and Birnbaumer, L. (1994). The amino termini of calcium channel β subunits set rates of inactivation independently of their effect on activation. Neuron 13, 1433-1438. 7. Qin, N., Olcese, R., Zhou, J., Cabello, O. A., Birnbaumer, L., and Stefani, E. (1996). Identification of a second region of the beta-subunit involved in regulation of calcium channel inactivation. Am. J Physiol 271, C1539-C1545. 8. Jones, L. P., Wei, S. K., and Yue, D. T. (1998). Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels. J Gen. Physiol 112, 125-143. 9. Cens, T., Restituito, S., Galas, S., and Charnet, P. (1999). Voltage and calcium use the same molecular determinants to inactivate calcium channels. J. Biol. Chem. 274, 54835490. 10. Sokolov, S., Weiss, R. G., Timin, E. N., and Hering, S. (2000). Modulation of slow inactivation in class A Ca2+ channels by beta- subunits. J. Physiol. (Lond. ) 527, 445-454. 11. Hering, S., Berjukow, S., Sokolov, S., Marksteiner, R., Weiss, R. G., Kraus, R., and Timin, E. N. (2000). Molecular determinants of inactivation in voltage-gated Ca2+ channels. J Physiol 528, 237-249. 12. Pragnell, M., De Waard, M., Mori, M., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994). Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature 368, 67-70. 13. Opatowsky, Y., Chen, C. C., Campbell, K. P., and Hirsch, J. A. (2004). Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain. Neuron 42, 387-399. 14. Chen, Y. H., Li, M. H., Zhang, Y., He, L. L., Yamada, Y., Fitzmaurice, A., Shen, Y., Zhang, H., Tong, L., and Yang, J. (2004). Structural basis of the alpha1-beta subunit interaction of voltage-gated Ca2+ channels. Nature 429, 675-680. 15. Van Petegem F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004). Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature 429, 671-675.
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16. Williams, M. E., Marubio, L. M., Deal, C. R., Hans, M., Brust, P. F., Philipson, L. H., Miller, R. J., Johnson, E. C., Harpold, M. M., and Ellis, S. B. (1994). Structure and functional characterization of neuronal alpha 1E calcium channel subtypes. J. Biol. Chem. 269, 22347-22357. 17. Qin, N., Platano, D., Olcese, R., Costantin, J. L., Stefani, E., and Birnbaumer, L. (1998) Unique regulatory properties of the type 2a Ca2+ channel beta subunit caused by palmitoylation. Proc. Natl. Acad. Sci. USA 95, 4690-4695. 18. Chien, A. J., Carr, K. M., Shirokov, R. E., Rios, E., and Hosey, M. M. (1996). Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function. J. Biol. Chem. 271, 26465-26468. 19. Restituito, S., Cens, T., Barrere, C., Geib, S., Galas, S., De Waard, M., and Charnet, P. (2000). The β2a subunit is a molecular groom for the Ca2+ channel inactivation gate. J. Neurosci. 20, 9046-9052. 20. Hurley, J. H., Cahill, A. L., Currie, K. P., and Fox, A. P. (2000). The role of dynamic palmitoylation in Ca2+ channel inactivation. Proc. Natl. Acad. Sci. USA 97, 9293-9298. 21. Stotz, S. C., Jarvis, S. E., and Zamponi, G. W. (2004). Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels. J. Physiol 554, 263-273. 22. Richards, M. W., Leroy, J., Pratt, W. S., and Dolphin, A. C. (2007). The HOOK-Domain Between the SH3- and the GK-Domains of CaVß Subunits Contains Key Determinants Controlling Calcium Channel Inactivation. Channels 1, 92-101. 23. Stotz, S. C., Barr, W., McRory, J. E., Chen, L., Jarvis, S. E., and Zamponi, G. W. (2004). Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit. J. Biol. Chem. 279, 3793-3800. 24. Berrou, L., Klein, H., Bernatchez, G., and Parent, L. (2002). A specific tryptophan in the III linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels. Biophys. J. 83, 1429-1442. 25. Gonzalez-Gutierrez, G., Miranda-Laferte, E., Neely, A., and Hidalgo, P. (2007). The Src Homology 3 Domain of the beta-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction. J Biol Chem 282, 2156-2162. 26. Gonzalez-Gutierrez, G., Miranda-Laferte, E., Naranjo, D., Hidalgo, P., and Neely, A. (2008) Mutations of Nonconserved Residues within the Calcium Channel α1-interaction Domain Inhibit ß-Subunit Potentiation. J. Gen. Physiol. 132, 383-395 27. Hidalgo, P., Gonzalez-Gutierrez, G., Garcia-Olivares, J., and Neely, A. (2006). The alpha 1-beta subunit interaction that modulates calcium channel activity is reversible and requires a competent alpha -interaction domain. J. Biol. Chem. 281, 24104-24110. 28. Schneider, T., Wei, X., Olcese, R., Costantin, J. L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., Crawford, G. D., and et al (1994). Molecular analysis and functional expression of the human type E neuronal Ca2+ channel alpha 1 subunit. Receptor and channels 2, 255-270 29. Gonzalez-Gutierrez, G., Miranda-Laferte, E., Nothmann, D., Schmidt, S., Neely, A., and Hidalgo, P. (2008). The guanylate kinase domain of the ß-subunit of voltage-gated calcium channels suffices to modulate gating. Proc. Natl. Acad. Sci. U. S A 105, 14198-14203.
FOOTNOTES * This work was supported by grants from the Deutsche Forschung Gemeinschaft (DFG Hi 800/3-1 and 4-1) to PH, Fondo Nacional de Ciencia y Tecnología (FONDECYT 1090635 to AN, and the GermanChilean Scientific Cooperation Program DFG-CONICYT 2010 to PH and AN. S This article contains Supplemental Methods 2 The abbreviations used are: HVA, high-voltage activated channels; CDI, calcium-dependent inactivation; VDI, voltage-dependent inactivation; PBLK, polybasic linker segment; PSLK, polyserine linker segment; SH3, Src homology 3domain; GK, guanylate kinase domain; AID, α-interaction domain; V1/2, voltage to reach 50% of channel activation; T0.5, time to reach 50% of peak current.
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30. Neely, A., Garcia-Olivares, J., Voswinkel, S., Horstkott, H., and Hidalgo, P. (2004). Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function. J. Biol. Chem. 279, 21689-21694. 31. Taglialatela, M., Toro, L., and Stefani, E. (1992). Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes. Biophys. J. 61, 78-82. 32. Miranda-Laferte, E., Gonzalez-Gutierrez, G., Schmidt, S., Zeug, A., Ponimaskin, E. G., Neely, A., and Hidalgo, P. (2011). Homodimerization of the Src homology 3 domain of the calcium channel beta-subunit drives dynamin-dependent endocytosis. J. Biol. Chem. 286, 22203-22210. 33. Olcese, R., Neely, A., Qin, N., Wei, X., Birnbaumer, L., and Stefani, E. (1996). Coupling between charge movement and pore opening in vertebrate neuronal alpha 1E calcium channels. J. Physiol. (Lond. ) 497, 675-686. 34. Parent, L., Schneider, T., Moore, C. P., and Talwar, D. (1997). Subunit regulation of the human brain alpha 1E calcium channel. J Membr. Biol. 160, 127-140. 35. Thierry Cens, Sophie Restituito, Sophie Restituito, and Pierre Charnet (2005). Role of ß Subunits in Voltage-Gated Calcium Channel Functions . In Voltage-Gated Calcium Channels, Gerald W Zamponi, editor. Kluwer Academic / Plenum Publishers, New York 36. Neely, A., Wei, X., Olcese, R., Birnbaumer, L., and Stefani, E. (1993). Potentiation by the β subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science 262, 575-578. 37. Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A. (2003). The voltage-dependent calcium channel beta subunit contains two stable interacting domains. J. Biol. Chem. 278, 52323-52332. 38. Ben-Tal, N., Honig, B., Peitzsch, R. M., Denisov, G., and McLaughlin, S. (1996). Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results. Biophys. J. 71, 561-575.
FIGURE LEGENDS
FIGURE 2. A short polybasic linker segment at the boundary of the GK domain slows down voltage-dependent inactivation of CaV2.3 channels. (A) Schematic representation of the domain structure of the β-subunit. The highly conserved SH3 and GK domains are flanked by three variable regions, N-terminal (NT), C-terminal (CT) and the linker region joining both domains (LK). Numbers denote amino acid position of the rat β2a isoform. The two cysteine residues at positions 3 and 4 (C3C4) that undergo palmitoylation in β2a are highlighted. We divided LK into two segments, a large poly-serine segment (PSLK), followed by a short polybasic segment (PBLK) at the boundary of GK domain (shown in red). Positively charged residues within PBLK are highlighted in red. (B) Representative current traces from Xenopus oocytes injected with CaV2.3 encoding cRNA and the indicated β-protein construct following the same protocol pulse as in Fig. 1B. β2a SH3-PBLK-GK consists of SH3 and GK domains joined by PBLK segment, while in β2a SH3-GK no linker segment is present between both domains. The insets show anti-β antibody Western Blot analysis of crude lysate from Xenopus oocytes injected with β2a SH3-PBLK-GK (left panel) or β2a SH3-GK (right panel), as described in Material and Methods; left lane, crude lysate from five non-injected oocytes; middle lane, crude lysate from five oocytes injected with the corresponding β-construct and right lane, purified β-construct before injection (80 ng loaded). (C) Bar plot of average T0.5 values for oocytes expressing CaV2.3 plus the indicated β-construct. Values are expressed as mean ± SEM. The dashed line corresponds to T0.5 for CaV2.3 channels expressed alone. (D) Voltage-dependent inactivation curves from oocytes injected with CaV2.3 encoding cRNA and the indicated β-protein measured and fitted as in Fig 1C. The curve for CaV2.3 alone is shown by a dotted line. FIGURE 3. β 2a SH3-PBLK-GK locates within the cytoplasm in mammalian cells. Confocal images of fluorescently labeled β2a, (β2a-YFP), β 2a SH3-PBLK-GK (β2a SH3-PBLK-GK-YFP) and β2a C3,4S (β2a C3,4S-YFP). All β2a derivatives were fused to YFP and expressed in tsA201 cells. Only β2a-YFP shows strong membrane localization while the others distribute within the cytoplasm.
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FIGURE 1. Co-injection of CaVα1-cRNA with β-proteins in Xenopus oocytes recapitulates CaV2.3 channel modulation. (A) Strategy for CaVα1 cRNA/β-protein co-injection. The cRNA encoding for CaVα1-subunit is injected into Xenopus laevis oocytes one hour before recombinant βsubunit injection. Electrophysiological recordings are performed five to six days later. (B) Representative current traces from oocytes injected with CaV2.3 encoding cRNA and the indicated β-protein during a 10 s pulse to 0 mV from a holding potential of -90 mV. Bar plot of average T0.5 values for the indicated channel subunit combination. Values are expressed as mean ± SEM. The dashed line corresponds to T0.5 for CaV2.3 channels expressed alone (in the absence of β-subunit). (C) Voltage-dependent inactivation curves from oocytes injected with CaV2.3 encoding cRNA and the indicated β-protein. The degree of inactivation was determined after a 10 s pre-pulse ranging from -120 mV to +30 mV in 15 mV increments from a holding potential of -90 mV followed by short deactivation pulse to -90 mV and a 0.5 s test pulse at 0 mV. Continuous lines correspond to double Boltzmann distributions that best fit the experimental data for the indicated subunit combination. For comparison the fit to the inactivation curve obtained with CaV2.3 expressed alone is shown by a dotted line. (D) Voltage-dependent activation curves from oocytes injected as in (C) obtained from tail currents measured at -40 mV after a 70 ms pulse ranging from -50 mV to +105 mV in 5 mV increments from a holding potential of -80 mV. Continuous lines correspond to the sum of two Boltzmann distributions that best fit the experimental data. The curve obtained with CaV2.3 expressed alone is shown by a dotted line.
FIGURE 4. Substitution of the basic residues by alanine within the polybasic linker segment eliminates slow-inactivation conferring phenotype of β 2a SH3-PBLK-GK. (A) Schematic representation of β2a SH3-PBLK-GK showing the amino acid sequence of native PBLK and PBLK with all positively charged residues (shown in red) substituted by alanine (β2a SH3-PBLKAla-GK). The inset shows Western Blot analysis using anti-β antibody of β2a SH3-PBLKAla-GK, as described in Material and Methods; left lane, crude lysate from five non-injected oocytes; middle lane, crude lysate from five oocytes injected with β2a SH3-PBLKAla-GK and right lane, purified β2a SH3-PBLKAla-GK before injection (80 ng loaded). (B) Representative current traces from oocytes injected with CaV2.3 encoding cRNA and β2a SH3-PBLKAla-GK following the same protocol as in Fig. 1B. For comparison, the current recording for CaV2.3/β2a SH3-PBLK-GK channel complexes from Fig. 2B is shown in light gray. C) Average T0.5 values for oocytes expressing CaV2.3/β2a SH3-PBLKAla-GK channel complexes. The dotted line denotes the average T0.5 for CaV2.3/β2a SH3-GK channels. T0.5 decreases from 0.98 ± 0.16 s for CaV2.3/β2a SH3PBLK-GK to 0.07 ± 0.01 s for CaV2.3/β2a SH3-PBLKAla-GK channel complexes. Values are expressed as mean ± SEM. D) Voltage-dependent inactivation curves from oocytes injected with CaV2.3 and β2a SH3PBLKAla-GK obtained as in Fig 1C. For comparison, steady state inactivation curve from oocytes expressing CaV2.3/β2a SH3-PBLK-GK shown in Fig. 2D is included (dotted line).
FIGURE 6. Palmitoylated N-terminus and the polybasic segment in β 2a regulate synergistically voltage-dependent inactivation of CaV2.3 channels. (A) Schematic representation of β2a lacking the polybasic linker segment (β2a ΔPBLK) and representative current trace from oocytes injected with CaV2.3 encoding cRNA and β2a ΔPBLK following the same protocol as in Fig. 1B. For comparison, traces from oocytes expressing CaV2.3/β2a (blue) and CaV2.3/β2a C3,4S (gray) channel complexes from Figs. 1B and 5A, respectively, are shown. Inset corresponds to Western Blot analysis using anti-β antibody, as described in Material and Methods: left lane, crude lysate from five non-injected oocytes; middle lane, crude lysate from five oocytes injected with β2a ΔPBLK and right lane, purified β2a ΔPBLK before injection (80 ng). B). Confocal image of tsA201 cells expressing β2a ΔPBLK fused to YFP, showing that removal of the PBLK does not impair membrane localization of the protein . C) Average T0.5 values for CaV2.3/β2a ΔPBLK channel complexes. The dashed lines denote average T0.5 for oocytes expressing CaV2.3/β2a (blue) and CaV2.3/β2aC3,4S (gray) channel complexes. Values are expressed as mean ± SEM. Removal of the polybasic linker in β2a decreases T0.5 from 1.93 ± 0.47 s for CaV2.3/β2a to 1.58 ± 0.17 s for
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FIGURE 5. The polyserine linker region but not the N- or C-terminal variable sequences mask the effect of the polybasic segment on voltage-dependent inactivation. (A) Schematic representation of β2a C3,4S double mutant and representative current traces from oocytes expressing CaV2.3/β2a C3,4S channel complexes following the same pulse protocol as in Fig. 1B. The inset shows Western Blot analysis using anti-β antibody, as described in Material and Methods: left lane, crude lysate from five non-injected Xenopus oocytes; middle lane, crude lysate from five oocytes injected with β2a C3,4S and right lane, purified β2a C3,4S before injection (80 ng). (B) Same as A but using β2a SH3-PBLK-GK that includes the N-terminal region of β2a C3,4S (β2a SH3-PBLK-GK + NTC3,4S). (C) Same as A but using β2a SH3-PBLKGK containing the C-terminal region of β2a (β2a SH3-PBLK-GK + CT). (D) Same as panel A but the β2a construct encompassing the complete linker region while missing the N-and C-terminal regions (β2a SH3LK-GK). (E) Bar plot of average T0.5 values from oocytes expressing CaV2.3 plus the indicated βderivative construct: NT, β2a SH3-PBLK-GK + NTC3,4S (black bar); CT, β2a SH3-PBLK-GK + CT (blue bar); LK, β2a SH3-LK-GK (red bar) and C3,4S, β2a C3,4S (green bar). The dashed line denotes average T0.5 for oocytes expressing CaV2.3/β2a SH3-PBLK-GK channel complexes. Values are expressed as mean ± SEM. F) Voltage-dependent inactivation curves from oocytes expressing the different subunit combinations shown in panel E. For comparison, the steady state inactivation curve from oocytes expressing CaV2.3/β2a SH3-PBLK-GK shown in Fig. 2D is included (dotted line).
CaV2.3/β2a ΔPBLK (t-test, p < 0.05) while substitution of the double cysteine motif results in faster T0.5 (0.34 ± 0.01 s for CaV2.3/β2a C3,4S). D) Voltage-dependent inactivation curves for the same subunit combinations shown in panel C. FIGURE 7. Model for modulation of voltage-dependent inactivation by β 2a subunit. Two molecular determinants of β-subunit control voltage-dependent inactivation; palmitoylated N-terminus and the polybasic linker segment (shown in red). The β-subunit binds through the GK domain to the conserved αinteraction domain in the CaVα1 subunit. According to current models, anchoring of β2a to the
membrane restricts the movement of the inactivation particle and, based on our present data, it exposes the polybasic segment that further inhibits VDI (left panel). In the absence of a palmitoylated N-terminus the region upstream the polybasic linker segment occludes its functional association with the inactivation machinery (right panel).
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TABLE 1 T0.5 Mean ± SEM 0.23 1.93 1.58 1.09 0.98 0.98 0.36 0.34 0.28 0.24 0.09 0.07
± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.47 0.17 0.07 0.10 0.16 0.05 0.06 0.02 0.05 0.01 0.01
-41.06 -36.43 -43.30 -56.08 -54.60 -48.96 -58.28 -57.17 -65.31 -63.19 -60.79 -65.03
± ± ± ± ± ± ± ± ± ± ± ±
1.09 2.32 0.95 3.28 4.92 2.18 1.93 2.49 0.99 2.64 3.01 2.19
% Ires Mean ± SEM 1.6% 16.6% 10.1% 3.0% 2.1% 3.0% 2.3% 1.2% 1.1% 0.5% 0.7% 1.1%
± ± ± ± ± ± ± ± ± ± ± ±
0.4% 2.7% 1.2% 0.9% 0.3% 0.9% 0.7% 0.3% 0.4% 0.2% 0.3% 0.3%
Table 1: Summary of Mean ± S.E.M for different parameters describing inactivation of the CaV2.3 channels expressed alone or with the indicated β-protein. The different constructs are described in the text and figures. Parameters from the CaV2.3 alone topping the list is followed by the different subunit combination in decreasing order for T0.5 that corresponds to the time for the current to return to 50% of its peak amplitude, evoked by a depolarizing pulse of 10 s to 0 mV. V1/2 corresponds to the voltage at which 50% of the channels are inactivated, estimated from the fit to a double Boltzmann distribution according to the following equation that also defines Ires. (1− I res ) I (V ) = I max * I res + zF z F − 1 (Vm−V2 ) − 2 (Vm−V2 ) RT RT 1+ e ⋅ 1 + e Where Imax is the current at peak, Ires is the non-inactivating current, F is the Faraday´s constant, R the Universal constant of gases, T is temperature (298 K), Vm is the membrane voltage. V1,V2, z1 and z2 are the parameter defining each Boltzmann component. V1/2 is then obtained by solving numerically I(V)=Imax/2. Ires corresponds to the fraction of channels that do not inactivate at extreme positive voltages.
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CaV2.3 CaV2.3/β2a CaV2.3/ β2a ∆PBLK CaV2.3/ β2a SH3-PBLK-GK+NTC3,4S CaV2.3/ β2a SH3-PBLK-GK+CT CaV2.3/ β2a SH3-PBLK-GK CaV2.3/ β2a C3,4 S to A CaV2.3/ β2a C3,4S CaV2.3/ β3xo CaV2.3/β2a SH3-LK-GK CaV2.3/ β2a SH3-GK CaV2.3/ β2a SH3-PBLKAla-GK
V1/2 Mean ± SEM
Figure 1
Oocyte injection α1-cRNA 1 hour
B
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Figure 2
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Figure 3
β2a SH3-PBLK-GK-YFP
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Figure 4
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CaV2.3/β2 a SH3-PBLK-GK CaV2.3/β2 a SH3-PBLKAla -GK
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Figure 5 A SH3
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Figure 6 A
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T0.5 (s)
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Figure 7 C 3C 4
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+++
SH3
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