Abstract
L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.
Highlights
As part of its ubiquitous role in controlling Ca2ϩ-dependent cellular processes, calmodulin (CaM)1 is an important regula
As a first step toward understanding the dual action of CaM, we have examined the contributions of individual amino acids of the IQ motif to Ca2ϩsensitive inactivation and facilitation of L-type channels expressed in Xenopus oocytes
Alanine Substitutions within the IQ Motif—Our previous analysis [25] was based on mutations at a single position, isoleucine 1624, the first of 12 residues in a consensus IQ motif in the C-terminal tail of the ␣1C subunit (Fig. 1A). Those experiments led to the conclusion that both Ca2ϩ-dependent inactivation and facilitation depend upon CaM binding to the IQ motif
Summary
CaM, calmodulin; bp, base pair; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid Na4 salt; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; HEDTA, Nhydroxyethylethylenediaminetriacetic acid. Mutations in the IQ motif that eliminate the CaM binding prevent both forms of modulation [25] All of these observations support the involvement of Ca2ϩ-dependent CaM binding to the L-type channel in both inactivation and facilitation, they raise the question of how both forms of autoregulation might arise from a single Ca2ϩ sensor [25]. This complexity exceeds that found in any other CaM-regulated ion channel so far. Both processes require CaM binding to the IQ motif, inactivation and facilitation depend on significantly different structural determinants within this motif
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