Architecture of Ca2+ channel pore-lining segments revealed by covalent modification of substituted cysteines

The cysteine accessibility method was used to explore calcium channel pore topology. Cysteine mutations were introduced into the SS1-SS2 segments of M otifs I-TV of the human cardiac L-type calcium channel, expressed in Xenopu s oocytes and the current block by methanethiosulfonate compounds was measu red. Our studies revealed that several consecutive mutants of motifs ZI and III are accessible to methanethiosulfonates, suggesting that these segment s exist as random coils. Motif I cysteine mutants exhibited an intermittent sensitivity to these compounds, providing evidence for a beta -sheet secon dary structure. Motif IV showed a periodic sensitivity, suggesting the pres ence of an cr-helix. These studies reveal that the SS1-SS2 segment repeat i n each motif have non-uniform secondary structures. Thus, the channel archi tecture evolves as a highly distorted 4-fold pore symmetry.