Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the alpha(1c) subunit

L-type Ca2+ channels are unusual in displaying two opposing forms of autore gulatory feedback, Ca2+- dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (C aM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of th e channel's cu,, subunit, Here we report the functional effects of an exten sive series of modifications of the IQ motif aimed at dissecting the struct ural determinants of the different forms of modulation. Although the combin ed substitution by alanine at five key positions (Ile(1624), Gln(1625) Phe( 1628), Arg(1629), and Lys(1630)) abolished all Ca2+ dependence, correspondi ng single alanine replacements behaved similarly to the wild-type channel ( 77wt) in four of five cases. The mutant I1624A stood out in displaying litt le or no Ca2+-dependent inactivation, but clear Ca2+- and frequency-depende nt 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 experime nts. Replacement of Ile1624 by 13 other amino acids produced graded and dis tinct patterns of change in the two forms of modulation. The extent of Ca2-dependent facilitation was monotonically correlated with the affinity of C aM for the mutant IQ motif, determined in peptide binding experiments in vi tro. Ca2+-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic si de chain at position 1624. Abolition of Ca2+-dependent modulation by IQ mot if modifications mimicked and occluded the effects of overexpressing a domi nant-negative CaM mutant.