Fast Inactivation of Voltage-dependent Calcium Channels

Abstract

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428 -22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type ␣ 1C and ␣ 1E calcium channels. Substitution of the II S6 and/or III S6 segments of ␣ 1E into the ␣ 1C backbone resulted in rapid inactivation rates that closely approximated those of wild type ␣ 1E channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type ␣ 1E channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of ␣ 1E S6 segments of domains II, III, or II؉III with those of ␣ 1C was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of ␣ 1E were concomitantly replaced with ␣ 1C sequence could inactivation be abolished. Conversely, introduction of the ␣ 1E domain I-II linker sequence into ␣ 1C conferred ␣ 1E -like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.Calcium entry through voltage-dependent calcium channels is important for a range of cellular processes, including neurotransmitter release and activation of Ca 2ϩ -dependent enzymes. Molecular cloning has identified the primary structures of at least 9 different neuronal Ca 2ϩ channel ␣ 1 subunits (termed ␣ 1A through ␣ 1I (1-15)) that encode the previously identified native L-, P-, N, -Q-, T-, and R-types (for review, see Refs. 16 and 17). Calcium channels, like many other voltage-dependent ion channels, undergo a series of conformational changes in response to voltage, resulting in their opening, closing, and inactivation. Voltage-dependent inactivation of calcium channels is an important intrinsic process that prevents the breakdown of the calcium gradient as well as excessive calcium entry that is toxic to most cells (18 -20). In addition, many pharmacological agents interact predominantly with inactivated channels (21, 22). Unlike sodium (23, 24) and potassium (25-27) channels, the mechanisms that govern calcium channel voltagedependent inactivation are not fully understood. Although a number of structural moieties of the calcium channel ␣ 1 subunit have been implicated in being important in fast calcium channel inactivation (7, 22, 28 -30, 32, 33), the detailed mechanism underlying the inactivation process remain unknown, and there have been few systematic attempts to resolve this issue. By creating a series o...

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

“…8). Although highly speculative, the model integrates related structure-function results from a number of different laboratories that point to the ␤ subunit D domain interaction with the ␣ 1 subunit I-II linker as a key determinant of Ca 2ϩ channel gating properties (Herlitze et al, 1997;Bourinet et al, 1999;Stotz et al, 2000;Berrou et al, 2001). Moreover, it incorporates results showing that regulation of activation and inactivation are separable functions of ␤ subunits (Olcese et al, 1994).…”

Amino Acids in Segment IVS6 and β-Subunit Interaction Support Distinct Conformational Changes during Cav2.1 Inactivation

“…Under this scenario, IVS6 residues would form part of a receptor for a ␤ 2a -modulated inactivation gate or lid interacting from the intracellular side of the channel pore (see Ref. 46 for a proposed hinged lid mechanism of Ca v 2.3 involving the I-II linker and segments IIS6 and IIIS6; see also Ref. 47).…”

The Interaction between the I-II Loop and the III-IV Loop of Cav2.1 Contributes to Voltage-dependent Inactivation in a β-Dependent Manner

“…, several studies have postulated that the I-II loop may act as an inactivation particle by physically occluding the pore (7,27,28). Although this hypothesis may seem attractive, the receptor site for such an inactivation particle has not been identified.…”