Identification of Inactivation Determinants in the Domain IIS6 Region of High Voltage-activated Calcium Channels

Abstract

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (␣ 1E ) calcium channels to slowly inactivating L-type (␣ 1C ) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, ␣ 1C and ␣ 1E channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of ␣ 1C with the corresponding ␣ 1E residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated ␣ 1C inactivation could still be antagonized following coexpression of the rat ␤ 2a subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.

Emergence of a R-Type Ca 2+ Channel (Ca V 2.3) Contributes to Cerebral Artery Constriction After Subarachnoid Hemorrhage

“…Further, 1 makes a greater contribution to current decay in cerebral artery myocytes isolated from SAH compared with control animals ( Figure 3D). These data demonstrate a fundamental change in the properties of VDCC currents, suggesting the possibility that a second, more rapidly inactivating, high voltage-activated Ca 2ϩ channel (eg, R-type 20 ) may contribute to enhanced VDCC currents in cerebral artery myocytes after SAH.…”

ACACNA1Fmutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation

“…Residue 745 is located toward the intracellular end of transmembrane segment IIS6 (44). Amino acids in the S6 segment have previously been implicated in the gating of voltagegated ion channels (44)(45)(46)(47)(48)(49). Activation of voltage-gated ion channels is commonly described by a three-state model ) and I745T (filled circles) channels.…”

Trafficking of L-type Calcium Channels Mediated by the Postsynaptic Scaffolding Protein AKAP79

“…A similar procedure was used to fuse AKAP79 or AKAP79⌬388 with a c-Myc epitope. Deletion of the PP motif on the ␣ 1C II-III loop (amino acids 854 to 864) was created by overlapping PCR, using as template a wild-type ␣ 1C cDNA engineered to contain two unique silent restriction sites (MluI and SpeI) flanking the II-III loop region (18 (19). When AKAP79 was coexpressed with the Ca 2ϩ channel subunits, we used a ratio of 1 (AKAP), 3 (Ca 2ϩ channel mix).…”