Cross talk between β subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV2.1 channel steady‐state inactivation
S. A. Serra et al.
Laboratori de Fisiologia Molecular Departament de Ciències Experimentals i de la Salut Universitat Pompeu Fabra Barcelona Spain
*CorrespondenceJosé M. Fernandez‐Fernández, Laboratory of Molecular Physiology, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, Barcelona 08003, Spain.
Tel: 34 93 3160854
Fax: 34 93 3160901
E‐mail: jmanuel.fernandez@upf.edu
Abstract
Modulation of CaV2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII‐III) of the CaV2.1 pore‐forming α 1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca2+ entry pathway, and by modulating the voltage dependence of channel steady‐state inactivation. Ca2+ influx through CaV2.1 also promotes channel inactivation. This process seems to involve Ca2+‐calmodulin interaction with two adjacent sites in the α 1A carboxyl tail (C‐tail) (the IQ‐like motif and the Calmodulin‐Binding Domain (CBD) site), and contributes to long‐term potentiation and spatial learning and memory. Besides, binding of regulatory β subunits to the α interaction domain (AID) at the first intracellular loop (LI‐II) of α 1A determines the degree of channel inactivation by both voltage and Ca2+. Here, we explore the cross talk between β subunits, Ca2+, and syntaxin‐1A‐modulated CaV2.1 inactivation, highlighting the α 1A domains involved in such process. β 3‐containing CaV2.1 channels show syntaxin‐1A‐modulated but no Ca2+‐dependent steady‐state inactivation. Conversely, β 2a‐containing CaV2.1 channels show Ca2+‐dependent but not syntaxin‐1A‐modulated steady‐state inactivation. A LI‐II deletion confers Ca2+‐dependent inactivation and prevents modulation by syntaxin‐1A in β 3‐containing CaV2.1 channels. Mutation of the IQ‐like motif, unlike CBD deletion, abolishes Ca2+‐dependent inactivation and confers modulation by syntaxin‐1A in β 2a‐containing CaV2.1 channels. Altogether, these results suggest that LI‐II structural modifications determine the regulation of CaV2.1 steady‐state inactivation either by Ca2+ or by SNAREs but not by both.
Article notes
S. A. Serra , G. G. Gené , X. Elorza‐Vidal , J. M. Fernández‐Fernández . Cross talk between β subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV2.1 channel steady‐state inactivation. Physiol Rep, 6 (2), 2018, e13557, https://doi.org/10.14814/phy2.13557 10.14814/phy2.13557PMC578971929380539
Footnote Group
Boxed Text
- The functional interaction between presynaptic voltage‐gated Ca2+ channels (CaV2.x) and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery, optimizes neurotransmitter‐mediated interneuronal communication.
- Alteration of such functional interaction has clinical relevance in the context of neurological disorders such as ataxia and migraine.
- Regardless of the important anchoring function of a specific CaV2.x region (the synprint site) in the CaV2.x‐SNAREs interaction, the involvement of other channel domains has been proposed.
- By combining heterologous expression in HEK 293 cells, whole‐cell patch‐clamp and site‐directed mutagenesis, we show that CaV2.1‐SNAREs functional interaction entails CaV2.1 molecular determinants beyond the synprint site, including the first intracellular loop and the carboxyl tail, and their physical interaction with regulatory β subunits and the Ca2+‐calmodulin complex, respectively.
- Altogether help us better understand the molecular machinery that initiates and regulates vesicles fusion with the presynaptic plasma membrane to trigger chemical neurotransmission.
Introduction
Ca2+ entry through the high‐voltage‐activated (HVA) CaV2.x channels (mainly CaV2.1 [P/Q‐type] channels) into presynaptic nerve terminals supports a transient Ca2+ microdomain that is essential for synaptic exocytosis leading to the fast release of classical neurotransmitters (Catterall 2011). To ensure fast and efficient neurotransmitter release, the vesicle‐docking/release machinery must be located near the pathway of Ca2+ entry. In many cases, this close localization is achieved by direct interaction of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins with the Ca2+ channel pore‐forming α 1 subunit, which consists of four repeated domains (I‐IV) each containing six transmembrane regions (S1–S6) with a voltage sensor (S1–S4) and a pore region (S5, P‐loop, and S6). Indeed, syntaxin‐1A/1B, SNAP‐25, and synaptotagmin‐1 specifically interact with CaV2.1 and CaV2.2 channels by binding to a synaptic protein interaction site (synprint) located within the intracellular loop connecting domains II and III (LII‐III) of the channels (Sheng et al. 1994, 1997; Rettig et al. 1996; Kim and Catterall 1997; Jarvis et al. 2002) (Fig. 1A). Furthermore, it has been suggested that exocytosis is activated even before Ca2+ entry, by conformational changes triggered during Ca2+ binding at the open CaV channel pore, which are transmitted from the channel to specific residues of CaV‐interacting SNARE proteins, accounting for the rapid time frame of evoked release (Atlas 2013; Bachnoff et al. 2013). Whichever the case, perhaps an equally important consequence of SNARE protein interaction with the Ca2+ channel is the modulation of presynaptic Ca2+ channel activity, thus fine‐tuning the amount of Ca2+ that binds to the pore, enters the synaptic terminal, and determines synaptic transmission strength. Specifically, the binding of syntaxin‐1A and SNAP‐25 to CaV2.1 and CaV2.2 α 1 subunits shifts the voltage dependence of steady‐state inactivation toward more negative membrane potentials following trains of brief depolarizing pulses to reduce channel availability, without affecting channel activation properties (Bezprozvanny et al. 1995; Zhong et al. 1999). Such inhibition is reverted, and channel activity fully restored, by synaptotagmin (Zhong et al. 1999). Thus, CaV2.x‐SNAREs interaction seems to optimize neurotransmission by favoring Ca2+ entry through channels presenting docked synaptic vesicles. Accordingly, the disruption of such functional interaction compromises not just vesicle exocytosis in vitro (Mochida et al. 2003; Harkins et al. 2004), but also synaptic transmission and the SNARE‐mediated inhibitory modulation of CaV2.x channels in vivo (Mochida et al. 1996; Rettig et al. 1997; Zamponi 2003; Keith et al. 2007). Moreover, human α 1A mutations impairing the functional interaction between CaV2.1 channels and SNARE proteins have clinical relevance in the context of ataxia and the phenotypic expression of both migraine with aura and hemiplegic migraine (Cricchi et al. 2007; Serra et al. 2010; Condliffe et al. 2013).
Beyond the important anchoring function of the synprint site in the SNARE‐mediated modulation of CaV2.x channel gating, the involvement of other molecular domains has been proposed. Hence, deletions within the LII‐III intracellular loop of the CaV2.2 α 1B channel subunit that completely eliminate the synprint site reduce but not abolish channel modulation by syntaxin, and syntaxin mutations that have no effect on binding affinity to α 1B‐synprint prevent the SNARE‐mediated regulation of CaV2.2 channel inactivation (Bezprozvanny et al. 2000). Besides, the A454T mutation (placed in the intracellular loop connecting domains I and II (LI‐II) of the CaV2.1 α 1A channel subunit, and associated to both early‐onset progressive ataxia (Cricchi et al. 2007) and the relief of migraine aura symptoms (Serra et al. 2010)) prevents the negative modulation of CaV2.1 channels by SNARE proteins and decreases channel coupling to exocytosis, thus revealing the importance of LI‐II structural integrity in the CaV2.1‐SNAREs functional interaction (Serra et al. 2010).
The molecular mechanism by which LI‐II influences CaV2.1‐SNAREs functional interaction is unknown. However, it is well established that LI‐II plays a determinant role in the regulation of CaV2.1 channel activity (Buraei and Yang 2010). In this sense, it has been suggested that conformational changes induced at LI‐II by the binding of functionally different regulatory β subunits not only determine the degree of voltage‐dependent inactivation but also the extent of a Ca2+‐dependent inactivation component (mediated by the binding of Ca2+‐calmodulin to two adjacent sites in the carboxyl tail [C‐tail] of the α 1A subunit: the IQ‐like motif and the Calmodulin‐Binding Domain [CBD] site) (Lee et al. 2000; DeMaria et al. 2001; Cens et al. 2006) (Fig. 1A). Interestingly, disruption of CaV2.1 modulation by calmodulin and related Ca2+ sensor proteins by mutation of the IQ‐like motif has been reported to impair long‐term potentiation and spatial learning and memory in mice (Nanoua et al. 2016).
Altogether, it draws a complex scenario in which CaV2.1 inactivation is produced by LI‐II and modulated by: β channel subunits interacting with LI‐II, SNARE proteins binding to the synprint site at the LII‐III but requiring the integrity of LI‐II, and Ca2+‐calmodulin attached to the C‐tail of the α 1A subunit.
To better understand the role of LI‐II in CaV2.1‐SNAREs functional interaction, we analyzed the modulation of CaV2.1 inactivation by syntaxin‐1A under intermediate and high Ca2+‐buffering conditions, in the presence of functionally different regulatory β subunits (β 2a or β 3) and distinct human α1A constructs containing either a LI‐II deletion around the A454 residue, mutations in the IQ‐like region, or a CBD deletion. Our results reveal a cross talk between different pathways involved in the modulation of CaV2.1 inactivation, showing that regulation by syntaxin‐1A of the human CaV2.1 channel activity requires both the integrity of α1A LI‐II and the lack of a Ca2+‐dependent component in the channel steady‐state inactivation.
Methods
cDNA constructs and site‐directed mutagenesis
cDNA of the human voltage‐gated Ca2+ (CaV2.1) channel α1A subunit (originally cloned into a pCMV vector) was a gift from Professor J. Striessnig (University of Innsbruck, Austria). cDNAs of the rabbit α 2 δ and rat β 3 and β 2a regulatory subunits, and syntaxin‐1A (subcloned into a pcDNA3 expression vector) were gifts from Dr. L. Birnbaumer (National Institutes of Health, North Carolina, USA) and Dr. J. Blasi (Universitat de Barcelona, Spain). CaV2.1 α 1A mutant subunits (ΔLI‐II451–457, IM/EE1964;1965, ΔCBD2020‐end) were generated using site‐directed mutagenesis (GenScript Corporation, Piscatway, NJ). All cDNA clones used in this study were sequenced in full to confirm their integrity.
Heterologous expression and electrophysiology
HEK 293 cells were transfected using a linear polyethylenimine (PEI) derivative, the polycation ExGen500 (Fermentas Inc., Hanover, Maryland, USA) as previously reported (eight equivalents PEI/3.3 μg DNA/dish) (Serra et al. 2010). For transfection, α1A (wild‐type [WT] or mutants), β 3 or β 2a, α 2 δ, and EGFP (transfection marker) cDNA constructs were used at a ratio of 1:1:1:0.3. When required, syntaxin‐1A was also cotransfected at the same ratio as CaV2.1 channel subunit cDNAs. Electrophysiological recordings were obtained from EGFP‐positive cells 24–48 h after transfection at room temperature (22–24°C).
Ca2+ currents (ICa 2+) through WT or mutant CaV2.1 channels containing β 3 or β 2a regulatory subunits were recorded in the whole‐cell configuration of the patch‐clamp technique, using a D‐6100 Darmstadt amplifier (List Medical, Germany). Pipettes had a resistance of 2–3 MΩ when filled with a solution containing (in mmol/L): 140 CsCl, 1 EGTA (intermediate Ca2+‐buffering condition) or 10 BAPTA (high Ca2+‐buffering condition), 4 Na2ATP, 0.1 Na3GTP, and 10 Hepes (pH 7.2–7.3 and 290–300 mOsmol/L). The external solution contained (in mmol/L): 140 tetraethylammonium‐Cl (TEACl), 3 CsCl, 2.5 CaCl2, 1.2 MgCl2, 10 Hepes, and 10 D‐glucose (pH 7.4 and 300–310 mOsmol/L). Previous work demonstrates that high levels of intracellular Ca2+ chelators (e.g., 10 mmol/L EGTA or 10 mmol/L BAPTA) impair CaV2.1 inactivation in a similar way as when replacing extracellular Ca2+ by Ba2+ (Lee et al. 2000). Such observation strongly suggest that chelator effect is due to Ca2+ buffering, ruling out any unwanted direct action of the Ca2+ chelator itself on channel inactivation. pClamp8 software (Molecular Devices, USA) was used for pulse generation, data acquisition, and subsequent analysis.
Steady‐state inactivation was estimated by measuring peak Ca2+ currents in response to a 50 ms (or 10 ms, when using the α 1A IM/EE mutant subunit) depolarizing test pulse (to +20 mV) from a holding of −80 mV, following 30‐sec steps to various holding potentials (conditioning pulses) between −80 and +20 mV (Fig. 1A). Between the 30‐sec conditioning depolarizations and the test pulse we employed a 20‐msec interpulse to the holding potential, which does not allow detectable recovery from inactivation of CaV2.x channels (Degtiar et al. 2000). This kind of protocol has been reported to detect significant increase in CaV2.x steady‐state inactivation induced by syntaxin‐1A (i.e., a left shift of V1/2 inact to more negative voltages by ~6 mV) (Degtiar et al. 2000). On the contrary, when using shorter (few seconds) conditioning pulses, the influence of syntaxin‐1A on CaV2.x channel gating was barely detectable (Degtiar et al. 2000). These results are consistent with the action of SNAREs on slow rather than fast channel inactivation (Degtiar et al. 2000). As described in detail previously (Serra et al. 2010), normalized I Ca 2+ persistent currents were fitted to the following Boltzmann equation in order to obtain half‐maximal voltage (V 1/2 inact) and slope factor (k inact) for steady‐state inactivation:
Statistics
Data are presented as the means ± SEM, and n represents the number of cells recorded for each experimental condition. Statistical significance was tested using one‐way Analysis of Variance (ANOVA) followed by a Bonferroni post hoc test. Differences were considered significant if P < 0.05. All statistical comparisons were performed using the GraphPad Instat software. All data are sampled from Gaussian (normal) distributions (tested using the method Kolmogorov and Smirnov).
Results
The impact of the SNARE protein syntaxin‐1A on the steady‐state inactivation of Ca2+ currents (ICa 2+) through wild‐type (WT) CaV2.1 channel containing the regulatory β 3 subunit (WTβ 3), was measured at intermediate (1 mmol/L EGTA) and high (10 mmol/L BAPTA) intracellular Ca2+‐buffering conditions to evaluate its calcium dependency. In both conditions, syntaxin‐1A expression favored channel steady‐state inactivation, as indicated by a significant left shift of V1/2 inact to more negative voltages (by ~5–9 mV) (Fig. 2B–E; Table 1). It must be noted that steady‐state inactivation of WTβ 3 channels was poorly dependent on intracellular Ca2+ concentration, as not significant differences were found when comparing ICa 2+ V1/2 inact values obtained at intermediate and high intracellular Ca2+‐buffering conditions in the absence of syntaxin‐1A (Fig. 2B and C (left panels), D and E (open circles); Table 1).
Interestingly, the introduction of a small deletion around the A454 residue at the first intracellular loop of the pore‐forming α1A subunit (ΔLI‐II451–457) (Fig. 1) made the steady‐state inactivation of β 3‐containing CaV2.1channels (ΔLI‐IIβ 3) Ca2+‐dependent. On one hand, ICa 2+ inactivation was reduced (with a significant ~8 mV right shift in the V1/2 inact) by increasing the buffering of intracellular Ca2+ (Fig. 3A and B (left panels), C and D (open circles); Table 1). On the other hand, such α1A LI‐II deletion removed the modulatory action of syntaxin‐1A on the steady‐state inactivation of CaV2.1 channels containing β 3 at intermediate Ca2+‐buffering condition (Fig. 3A, C [left panel], and D; Table 1), when Ca2+ entry through the channel promotes inactivation. The effect of syntaxin‐1A on the inactivation of the ΔLI‐IIβ 3 channel was recovered (V1/2 inact was significantly left‐shifted by ~6 mV) by increasing intracellular Ca2+ buffering to abrogate the novel LI‐II deletion‐induced Ca2+‐dependent component of inactivation (Fig. 3B and C [right panel], D; Table 1).
As widely reported before (for a review see Buraei and Yang 2010), CaV2.1 inactivation was substantially right‐shifted to more depolarized potentials for β 2a‐containing than for β 3‐containing channels (Fig. 4 vs. Fig. 2; Table 1). Under this condition, unlike WTβ 3 channels, the steady‐state inactivation of ICa 2+ through the β 2a‐containing WT CaV2.1 channel (WTβ 2a) presented a Ca2+‐dependent component (Fig. 4) that is not affected by the deletion in the first intracellular loop of the α1A subunit (ΔLI‐II451–457) (Fig. 5). Thus, V1/2 inact was significantly shifted to less negative values for both WTβ 2a and ΔLI‐IIβ 2a channels (by ~9 mV) when increasing intracellular Ca2+ buffering (Fig. 4A and B (left panels), C and D (open circles); Fig. 5A and B (left panels), C and D (open circles); Table 1). Accordingly, such right shift in the voltage dependence of inactivation disappeared once the β 2a‐containing CaV2.1 channel was rendered insensitive to Ca2+ by the introduction of a double mutation (IM to EE) at the calmodulin‐binding IQ‐like motif (Fig. 6A and B [left panels], C and D [open circles]; Table 1). Modulation by syntaxin‐1A of WTβ 2a and ΔLI‐IIβ 2a ICa 2+ steady‐state inactivation was occluded when the Ca2+‐dependent component was present (Fig. 4A and C [left panel], D; Fig. 5A and C [left panel], D; Table 1). Syntaxin‐1A‐induced left shift of V1/2 inact (by ~5–6 mV) was only present when the Ca2+‐dependent component of the steady‐state inactivation of β 2a‐containing CaV2.1 channels was removed, either by increasing intracellular Ca2+ buffering (Fig. 4B and C [right panel], D; Fig. 5B and C [right panel], D; Table 1) or by introducing the IM/EE double mutation at the IQ‐like motif (Fig. 6A–D; Table 1).
Truncation of the α1A carboxyl tail, downstream the IQ‐like motif, to fully remove the distal calmodulin‐binding domain (CBD) site of the β 2a‐containing CaV2.1 channel (ΔCBDβ 2a) did not eliminate the Ca2+‐dependent component of ICa 2+ steady‐state inactivation, and V1/2 inact was still significantly shifted to less negative values (by ~9 mV) when increasing intracellular Ca2+ buffering (Fig. 7A and B (left panels), C and D (open circles); Table 1). At intermediate intracellular Ca2+ buffering, the presence of the Ca2+‐dependent component in the steady‐state inactivation of ΔCBDβ 2a channels hindered their modulation by syntaxin‐1A (Fig. 7A and C (left panel), D; Table 1), and the SNARE protein only shifted V1/2 inact to more negative potentials (by ~5 mV) under high intracellular Ca2+ buffering (Fig. 7B and C (right panel), D; Table 1).
Discussion
Taken together, our results bring to light a functional cross talk between three different signaling pathways regulating CaV2.1 channel steady‐state inactivation: (1) regulatory β subunits through their interaction with the α interaction domain (AID) located at the first intracellular loop (LI‐II) of the CaV2.1 pore‐forming α 1A subunit (Buraei and Yang 2010), (2) Ca2+‐calmodulin binding to the IQ‐like motif at the α 1A C‐tail (DeMaria et al. 2001; Cens et al. 2006; this report), and (3) syntaxin‐1A, quite possibly, via its binding to the synprint site at the intracellular loop between domains II and III (LII‐III) of α 1A (Sheng et al. 1994, 1997; Rettig et al. 1996; Kim and Catterall 1997; Jarvis et al. 2002).
As previously reported for fast inactivation (Lee et al. 2000), we observed a substantial Ca2+‐dependent component in the steady‐state inactivation of CaV2.1 only in the presence of the palmitoylated, membrane‐anchored β 2a subunit (which, contrary to other regulatory β subunits (such as β 1 or β 3), reduces voltage‐dependent inactivation (Birnbaumer et al. 1998)). In agreement with findings from DeMaria et al. (2001) on CaV2.1 fast inactivation, the Ca2+‐dependent component of the steady‐state slow inactivation required the Ca2+‐calmodulin‐binding IQ‐like motif, with no detectable role of the previously involved CBD site (Lee et al. 2000). Hence, CaV2.1 Ca2+‐dependent steady‐state inactivation was abolished by the introduction of the double mutation IM/EE at the IQ‐like motif, but unaffected by a truncation of the α1A C‐tail, downstream the IQ‐like motif, that fully removes the CBD site. Besides, the Ca2+‐dependent component of CaV2.1 steady‐state inactivation seems to depend also on specific conformational changes induced by the binding of the functionally different β subunits at the α 1A LI‐II. Thus, the introduction of a LI‐II deletion (ΔLI‐II451–457) downstream the AID, around the A454 residue (of relevance for the modulation of CaV2.1 inactivation by β subunits and SNAREs (Serra et al. 2010)), made Ca2+‐sensitive the steady‐state inactivation of β 3‐containing CaV2.1 channels. Such CaV2.1 LI‐II deletion affects a fragment of a poorly conserved LI‐II region of 13 amino acids that in the cardiac CaV1.2 channel can bind Ca2+‐calmodulin (Pitt et al. 2001) (Fig. 8). Still, removal of this CaV1.2 region (ΔLI‐II520–532) did not abolish the Ca2+‐dependent component of cardiac channel inactivation (Pitt et al. 2001). This result agrees with our observation that ΔLI‐II451–457 had no effect on the Ca2+‐dependent inactivation of CaV2.1 channels containing the β 2a subunit.
Interestingly, syntaxin‐1A was only able to modulate CaV2.1 steady‐state inactivation when the Ca2+‐dependent component was absent either because of the presence of β 3 in a channel formed by a α 1A subunit with unaltered LI‐II, or due to the removal of channel Ca2+‐sensitivity by high intracellular Ca2+ buffering or by mutation of the IQ‐like motif.
Whether the above described functional cross talk is due to a three‐dimensional rearrangement of the involved α 1A intracellular domains (i.e., LI‐II, LII‐III and C‐tail), and the subsequent alteration of the interaction pattern between them and/or with their interacting partners (regulatory β subunits, SNARE proteins, and the Ca2+‐calmodulin complex), remains to be elucidated. However, there is evidence that make this hypothesis plausible since it has been reported that N‐tail, intracellular loop between domains III and IV (LIII‐IV) and C‐tail regions of CaV2.x or CaV1.2 α 1 subunits modulate channel inactivation through direct and dynamic interactions with LI‐II, or indirectly via regulatory β subunits (Geib et al. 2002; Kim et al. 2004; Stotz et al. 2004). To date, there are no structural data regarding the whole CaV2.1 channel complex that allow us to confirm these physical interactions between α 1A cytoplasmic domains. Nevertheless, the cryo‐electron microscopy (cryo‐EM) structure of the rabbit CaV1.1 complex, containing the pore‐forming α 1S and the regulatory α 2 δ 1, β 1a, and γ subunits, has been recently resolved with high, near‐atomic (3.6 Å) resolution (Wu et al. 2016). The structural analysis provides an atomic model for a potentially inactivated state of the CaV1.1 channel. In relation to the three‐dimensional arrangement of α 1S intracellular domains, the structural data locate the AID motif at LI‐II packed in between the regulatory β 1a subunit and the voltage sensor of α 1S domain II, and shows the formation of a globular helical domain due to the interaction between LIII‐IV and the proximal C‐tail (upstream the IQ motif) (Wu et al. 2016). The substantial homology between rabbit α 1S and human α 1A subunits with regard to residues involved in such LIII‐IV/C‐tail physical interaction (Fig. 9) suggests a similar scenario for the CaV2.1 channel. Unfortunately, several cytoplasmic segments were not visible in the cryo‐EM structure of CaV1.1 α 1S subunit, and the structure of LI‐II upstream the AID, the whole LII‐III and the C‐tail after residue D1515 (including the IQ motif) could not be resolved (Wu et al. 2016). Therefore, there are no structural data available neither on the possible interaction of LI‐II with either LIII‐IV or the C‐tail, or on any interaction involving LII‐III.
Biochemical experiments with recombinant proteins in vitro strongly indicate that the synprint site, located at LII‐III of α 1A/B, serves an important anchoring function that may facilitate SNARE's modulation of CaV2.1 and CaV2.2 gating (Sheng et al. 1994, 1997; Rettig et al. 1996; Kim and Catterall 1997; Jarvis et al. 2002). Nonetheless, functional studies also suggest that the regulatory action of SNAREs might involve binding to other sites in the pore‐forming α 1 channel subunit, and LI‐II and the C‐tail regions have been proposed as candidates (Bezprozvanny et al. 2000; Serra et al. 2010). Supporting this idea, recent findings show that low voltage‐activated CaV3.x (T‐type) α 1 channel subunits, which do not contain the consensus synprint site, biochemically interact with syntaxin‐1A and SNAP‐25 at the carboxy‐terminal domain (Weiss et al. 2012). In particular, syntaxin‐1A binding to CaV3.x channels potently modulates channel gating in a similar way that found for CaV2.x channels (Weiss et al. 2012). Besides, CaV3.x‐SNAREs interaction also appears essential for T‐type channel‐triggered low‐threshold exocytosis (Weiss et al. 2012), thus providing a molecular mechanism for their coupling to neurotransmitter and hormone release in neurons and neuroendocrine cells near resting conditions or during mild stimulations (Carbone et al. 2014).
In conclusion, our data suggest that conformational modifications of α 1A LI‐II (due to the binding of a particular regulatory β subunit, mutation A454T (Serra et al. 2010), or deletion ΔLI‐II451–457) determine the modulation of CaV2.1 steady‐state inactivation either by Ca2+ or by SNAREs but not by both.
Conflict of Interest
The authors declare that no conflict of interests exists.
In memoriam
In memory of Gemma G. Genè, PhD (1977–2017).
Acknowledgments
We are grateful to Dr. J. Striessnig (University of Insbruck, Austria) for the gift of human CACNA1A cDNA and Dr. J. Blasi (Universitat de Barcelona, Spain) for providing syntaxin‐1A cDNA. We also thank Dr. L. Birnbaumer (National Institutes of Health, North Carolina, USA) for the gift of the cDNAs encoding rabbit α 2 δ, and rat β 2a and β 3 regulatory subunits, and Dr. F. Rubio‐Moscardó for excellent technical assistance.