A New β Subtype-specific Interaction in α1ASubunit Controls P/Q-type Ca2+ Channel Activation

Abstract

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...

Alternative Splicing of the β4Subunit Has α1Subunit Subtype-Specific Effects on Ca2+Channel Gating

“…Previous studies have shown that the D domain of the ␤ 4 subunit binds with high affinity to the ␣ 1A I-II linker (Pragnell et al, 1994) and that the E domain of the ␤ 4 subunit binds to both the N and C terminus of the ␣ 1A subunit (Walker et al, , 1999. As shown in Figure 8, this indicates that the D and E domains likely establish the N-to C-terminal orientation of the ␤ 4 subunit relative to the ␣ 1A subunit.…”

“…Row 2 shows that adding back the C terminus to ␤ 4 ⌬N⌬C (␤ 4 ⌬N) shifts ␣ 1A complexes to mode A 2 I 2 , whereas ␣ 1B complexes remain in mode A 1 I 1 . This could be explained by the ␣ 1A -␤ 4 C-terminal binding event described by Walker et al ( , 1999 causing the ␤ 4 D domain to be displaced in the C-terminal direction. Relative to mode A 1 I 1 , activation in mode A 2 I 2 is shallower and inactivation is steeper.…”

“…[The effects of ␤ 4 N-terminal alternative splicing on apparent gating charge (z value) are shown in Table 1. Note the (Pragnell et al, 1994) and ␤ 4 E domain with ␣ 1A N and C termini have been well documented (Walker et al, , 1999. Dashed arrow in the bottom diagram indicates the potential for a conformational change when the ␤ 4a N terminus is substituted for the ␤ 4b N terminus.…”

“…We next sought to determine whether the ␤ 4 N terminus could be acting in concert with the ␤ 4 C terminus to exert its effects on gating. Because previous studies had shown that the ␤ 4 C terminus binds directly to the ␣ 1A subunit (Walker et al, , 1999, it was of particular interest to determine whether the gating properties of ␣ 1A would change in comparison to ␣ 1B if the ␤ 4 C terminus were A, B, The ␣ 1A (BI-2) and ␣ 1B (⌬21) subunits used in these and subsequent experiments are those described by Mori et al (1991) and Pan and Lipscombe (2000), respectively. Currents were activated by 300 msec depolarizations to various test potentials (Ϫ40 to ϩ40 mV in 5 mV increments) from a holding potential of Ϫ80 mV.…”

Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca 2+ -channel β 4 subunit

“…Together with the clear detection of ␤ 4c subunit expressed in Xenopus oocytes on Western blots (data not shown) and the protein's capability of interacting with the loop I/II of the ␣ 1 subunit, these results indicate that ␤ 4c is a functional Ca 2ϩ -channel ␤ subunit. They also imply a physiological role of the secondary interaction sites localized in the carboxyl-terminal half of ␤ 4a in the regulation of the channel's voltage dependence and inactivation kinetics (22,23).…”