The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.

The cytoplasmic ␤ subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the ␣ 1 interaction domain (AID) in the I-II cytoplasmic loop of ␣ 1 and the ␤ interaction domain (BID) of the ␤ subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of ␣ 1A (BI-2) and the carboxyl terminus of ␤ 4 . Like the interaction identified previously (21) between the carboxyl termini of ␣ 1A and ␤ 4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on ␤ 4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of ␣ 1 is isoform-dependent, suggesting a role in the specificity of ␣ 1 -␤ pairing. Coexpression of ␤ 4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the ␣ 1A channel compared with ␤ 3 (not exhibiting this interaction). Replacing the amino terminus of ␣ 1A with that of ␣ 1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Qtype channels.Despite their functional diversity, high voltage-gated Ca 2ϩ channels have three subunit types in common (1, 2). The ␣ 1 , pore-forming component of the channel is associated with a cytoplasmic ␤ subunit of 52-78 kDa and a largely extracellular ␣ 2 ␦ component, anchored by a single transmembrane domain. These subunits are encoded by at least 7 ␣ 1 , 4 ␤, and 1 ␣ 2 ␦ genes, respectively, of which numerous splice variants exist (3).The ␤ subunit, when coexpressed with the ␣ 1 subunit, results in an increase in current density, alteration of the voltage dependence and kinetics of both inactivation and activation, and an increase in the number of recognition sites for channelspecific ligands (for review, see Refs. 4 and 5). These effects reflect not only conformational modulation but also an increase in the number of channels properly addressed to the cell surface, suggesting multiple roles for the ␤ subunit. Although the effects of ␤ are highly conserved, significant differences are seen depending on the combination of ␣ 1 and ␤ subunits studied. For example, the kinetics of inactivation shows a general trend of variation with ␤ subtype (6 -9), whereas a shift in the voltage dependence of inactivation has been reported only for non-L-type, A, B, and E (10 -12), and not L-type channels (13). ␤ subunits also seem to differ in the mechanism by which they become localized to the plasma membrane (14, 15), perhaps suggesting that they are differentially targeted. Finally, ␣ 1 and ␤ subtypes differ in their potential (based on sequence predictions) to be phosphorylated by various protein kinases. These factors together point to a functional explanation...

We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca v 2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca v 2.1. As they are known to affect inactivation, we also analyzed the effects of ␤ subunits on these interactions. In experiments in which the ␤ 4 subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the ␤ subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the ␤ subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.The influx of calcium through voltage-gated calcium channels controls a variety of cellular processes ranging from membrane excitability and synaptic efficacy to gene expression. Both the amplitude and the duration of the calcium influx shape the spatio-temporal efficacy of calcium signaling. A tight control of both processes is needed to avoid long term increases in intracellular calcium levels, which are cytotoxic to neurons. Although the control of calcium entry can be achieved in several ways, inactivation of voltage-gated calcium channels appears to represent a key molecular process. For instance, inactivation is considered as a candidate mechanism for synaptic depression (1, 2). Inasmuch as there are several calcium channel types, there are also several inactivation behaviors. L-type calcium channels inactivate slowly, whereas the neuronal N-, P/Q-, and R-type channels inactivate faster. These fundamental differences are linked to the pore-forming Ca v subunit, which contains the major molecular determinants for inactivation, although auxiliary subunits can play a regulatory function in this process.

Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as 'ER retention'). This study demonstrates that the I-II loop of Cavalpha2.1 contains multiple ER-retention determinants beside the beta subunit association domain. In addition, the I-II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I-II loop, the N- and C-termini of Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i). a correct folding of Cavalpha in order to mask several internal ER-retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants.