Structure-function Relationship of N-terminal Deletion Mutants of Cardiac L-type Calcium Channel ß1-subunits

Auxiliary s-subunits of L-type Ca2+ channel (L-VDCC) profoundly modulate properties of L-VDCC. Previously, we demonstrated that the N-terminus of s2-subunit serves as a length-dependent structural determinant of channel inactivation (Herzig et al., FASEB J. 2007). Here, we tested the role of the N-terminus of s1a-subunit. Three artificial s1a-subunit mutants with different N-terminus lengths, s1aN18, s1aN27 and s1aN51, were created. Their modulatory functions were investigated in recombinant L-VDCC and compared with the natural full-length isoform, termed s1aN60.In whole-cell patch-clamp measurements, we confirmed functional expression of all s1a-subunit isoforms by a marked increase of current density and a leftward shift of activation, as compared to control transfections without any s-subunit. No obvious differences were found among s1a-subunit isoforms. In contrast, shortenening of the N-terminus progressively decreased the rate and extent of time-dependent inactivation at all test voltages.Descriptive analysis of the single-channel data (e.g., peak ensemble average current, open probability, availablility) revealed similar parameters among s1a-subunit isoforms, except for small deviations with s1aN51. Strikingly, the extent of the inactivation of ensemble average currents followed the length of the N-terminus (s1aN60>s1aN51>s1aN27>s1aN18). For more detailed kinetic analysis, we performed Markov modeling using the scheme:C-C-C-C-C-OIc-Ic-Ic-Ic-Ic-Iowith the rate constants for C-C and Ic-Ic: alpha, beta; C-O and Ic-Io: alpha´, beta´; C-I and O-I: gamma, delta. Channel open probability, availability, and first-latency, open-time and closed-time histograms were well fitted simultaneously. We found significant linear correlation between the inactivation rates gamma and delta and the N-terminus length. The other parameters alpha, alpha´, beta, beta´) did not vary with the N-terminal length of the s1a-subunit.Our results demonstrate that inactivation is under length-dependent control of the N-terminus of L-VDCC s1-subunit. This could represent a general mechanism of s-subunit modulation.