The Journal of Neuroscience, June 7, 2006, 26(23):6117-6118; doi:10.1523/JNEUROSCI.1699-06.2006
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The Calcium Channel 
4a Subunit: A Scaffolding Protein Between Voltage-Gated Calcium Channel and Presynaptic Vesicle-Release Machinery?
Norbert Weiss1,2,3
1Institut National de la Santé et de la Recherche Médicale U607, Laboratoire Canaux Calciques, Fonctions et Pathologies, 38054 Grenoble Cedex 09, France, 2Commissariat à l'Energie Atomique, Grenoble, France, and 3Université Joseph Fourier, Grenoble, France
Neuronal voltage-gated calcium channels represent a major pathway for calcium entry into nerve termini, where they control neurotransmitter release. The channels are composed of a pore-forming subunit (Cav2.x) and auxiliary subunits (Cav
1–4, 
2
1–4 and 
1–8). The cytoplasmic Cav subunits belong to the membrane-associated guanylate kinase (GK) family of proteins and regulate trafficking to the plasma membrane and gating properties of Cav2.x channels. Their five domains (A–E), include hypervariable A, C, and E domains linked to the highly conserved Src homology 3 (SH3) (B) and GK (D) domains. The crystal structure of the core domains (B–D) of several subunits was recently elucidated (Chen et al., 2004
; Opatowsky et al., 2004; Van Petegem et al., 2004), but less is known about the structure of the A and E domains. A recent report published in The Journal of Neuroscience provides new information on the Cav4a subunit hypervariable A domain (Cav4a-A) (Vendel et al., 2006a). The authors previously showed that alternative splicing of the Cav4-A domain generates two distinct proteins, Cav4a and Cav4b (Helton and Horne, 2002), and they recently provided the solution structure of the Cav4a-A domain (Vendel et al., 2006b). Vendel et al. now address the expression pattern of the Cav4a splice variant and the functional importance of the Cav4a-A domain.
Using immunohistochemistry, the authors show that the Cav4a splice variant was expressed as punctuate structures throughout the molecular layer of the cerebellum [Vendel et al. (2006a), their Fig. 3D,E (http://www.jneurosci.org/cgi/content/full/26/10/2635/F3)]. In contrast, Cav4b was expressed in basket cells surrounding Purkinje cell bodies as well as in the Bergmann glia [Vendel et al. (2006a), their Fig. 3F (http://www.jneurosci.org/cgi/content/full/26/10/2635/F3)]. To determine the functional importance of the Cav4a-A domain, the authors performed two electrode voltage-clamp recording in Xenopus laevis oocytes expressing Cav2.1 channels (P/Q type currents) in combination with 2 and Cav4a subunits with (ABCDE) or without (BCDE) the N-terminal A domain. The Cav4a-A domain was not essential for the expression of the Cav2.1 channel at the plasma membrane and did not influence gating properties. Indeed, no difference was observed in current amplitude [Vendel et al. (2006a), their Fig. 2A (http://www.jneurosci.org/cgi/content/full/26/10/2635/F2)], nor in the voltage dependence of activation or inactivation [Vendel et al. (2006a), their Fig. 2C,D (http://www.jneurosci.org/cgi/content/full/26/10/2635/F2)]. These results are, however, in agreement with published studies (Bichet et al., 2000; Van Petegem et al., 2004) because (1) enhanced Cav2.x subunit trafficking to the plasma membrane occurs after binding of the Cav subunit to the channel (Bichet et al., 2000) via the interaction domain (AID)-binding pocket within the GK domain (Van Petegem et al., 2004), and (2) the molecular determinants by which the Cav subunit modulates channel gating remain unclear but are probably carried by the conserved core domains because all Cav subtypes are able to modulate the biophysical properties of the channel. However, it is more surprising that channel inactivation kinetics was not influenced by deletion of the Cav4a-A domain [Vendel et al. (2006a), their Fig. 2B (http://www.jneurosci.org/cgi/content/full/26/10/2635/F2)]. Indeed, it is known that a deletion of the N-terminal of Cav subunits results in a drastic slowing of channel inactivation kinetics (Olcese et al., 1994). It seems not to be the case for the Cav4a splice variant. Thus, further investigations will provide interesting structural information on how the N-terminal of Cav subunits controls channel inactivation.
Because the Cav4a-A domain was not a key determinant in the regulation of Cav2.1 channel gating, Vendel et al. looked for a role of this domain in protein–protein interactions. Using the yeast two-hybrid system, the authors screened a human cerebellum cDNA library with the Cav4a-A domain (amino acids 1–58). Synaptotagmin I (Syt I) (amino acids 95–337 including the entire C2A domain and a half of the C2B domain) as well as the microtubule-associated protein 1A (MAP1A) (amino acids 2508–2775 corresponding to the complete LC2 domain) interacted specifically with the N-terminal A domain of the Cav4a splice variant but not with the Cav4b-A domain [Vendel et al. (2006a), their Fig. 4B (http://www.jneurosci.org/cgi/content/full/26/10/2635/F4)]. The authors then focused their study on Syt I and confirmed its interaction with the Cav4a-A domain by in vitro pull-down experiments. The interaction did not occur in the presence of 10 mM Ca2+ and could also be disrupted by adding Ca2+ to the medium [Vendel et al. (2006a), their Fig. 5C (http://www.jneurosci.org/cgi/content/full/26/10/2635/F5)]. These data certainly represent the most important findings of this study.
In conclusion, Vendel et al. (2006a) provide evidence that alternative splicing of the N-terminal A domain of the Cav4 auxiliary subunit confers functions other than modulations of channel gating and trafficking. Because Cav4a splicing variant can bind synaptotagmin I, an important protein for presynaptic vesicle release, the subunit, could conceivably act as a scaffolding element to facilitate coupling of calcium signaling with neurotransmitter release (Fig. 1). However, the authors do not provide evidence that Cav4a can bind Cav2.1 channel and Syt I simultaneously. Pull-down experiments performed by preincubating the full-length Cav4a with the AID peptide (the molecular determinant of the Cav2.x channel, which interacts with the AID-binding pocket of the Cav subunit) before adding Syt I could partially answer this question. Finally, the fact that Cav4a–Syt I interaction is disrupted by Ca2+ is interesting. In this context, we speculate that at basal Ca2+ levels, Cav4a interacts both with Cav2.1 and Syt I to organize the vesicle-release machinery and that calcium entry into cells via voltage-gated calcium channels breaks this interaction, thus releasing the vesicle to allow fusion with the plasma membrane. FRET experiments using tagged Syt I and Cav4a in the presence of Cav2.1 channels before and during membrane depolarization might address such a possible mechanism.
Received April 20, 2006;
accepted April 27, 2006.
Footnotes
Review of Vendel et al. (http://www.jneurosci.org/cgi/content/full/26/10/2535)
Correspondence should be addressed to Norbert Weiss, Institut National de la Santé et de la Recherche Médicale U607, Laboratory Canaux Calciques, Fonctions et Pathologies, Commissariat à l'Energie Atomique/DRDC/Bâtiment C3, 17 Rue des Martyrs, 38054 Grenoble Cedex 09, France. Email: norbert.weiss{at}cea.fr
DOI:10.1523/JNEUROSCI.1699-06.2006
Copyright © 2006 Society for Neuroscience 0270-6474/06/266117-02$15.00/0
References
Bichet D, Cornet V, Geib S, Carlier E, Volsen S, Hoshi T, Mori Y, De Waard M (2000) The I-II loop of the Ca2+ Channel 1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit. Neuron 25:177–190.[CrossRef][Web of Science][Medline]
Chen YH, Li MH, Zhang Y, He LL, Yamada Y, Fitzmaurice A, Shen Y, Zhang H, Tong L, Yang J (2004) Structural basis of the 1- subunit interaction of voltage-gated Ca2+ channels. Nature 429:675–680.[CrossRef][Medline]
Helton TD, Horne WA (2002) Alterative splicing of the 4 subunit has 1 subtype-specific effects on Ca2+ channel gating. J Neurosci 22:1573–1582.[Abstract/Free Full Text]
Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, Birnbaumer L (1994) The amino terminus of a calcium channel subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron 13:1433–1438.[CrossRef][Web of Science][Medline]
Opatowsky Y, Chen CC, Campbell KP, Hirsch JA (2004) Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the 1 interaction domain. Neuron 42:387–399.[CrossRef][Web of Science][Medline]
Van Petegem F, Clark KA, Chatelain FC, Minor DL (2004) Structure of a complex between a voltage-gated calcium channel b-subunit and an alpha-subunit domain. Nature 429:671–675.[CrossRef][Medline]
Vendel AC, Terry MD, Striegel AR, Iverson NM, Leuranguer V, Rithner CD, Lyons BA, Pickard GE, Tobet SA, Horne WA (2006a) Alternative splicing of the voltage-gated Ca2+ channel 4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex. J Neurosci 26:2635–2644.[Abstract/Free Full Text]
Vendel AC, Rithner CD, Lyons BA, Horne WA (2006b) Solution structure of the N-terminal A domain of the human voltage-gated Ca2+ channel 4a subunit. Protein Sci 15:378–383.[CrossRef][Web of Science][Medline]
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