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 Motifs I-IV of the human cardiac L-type calcium channel, expressed in Xenopusoocytes and the current block by methanethiosulfonate compounds was measured. Our studies revealed that several consecutive mutants of motifs II and III are accessible to methanethiosulfonates, suggesting that these segments exist as random coils. Motif I cysteine mutants exhibited an intermittent sensitivity to these compounds, providing evidence for a β-sheet secondary structure. Motif IV showed a periodic sensitivity, suggesting the presence of an α-helix. These studies reveal that the SS1-SS2 segment repeat in each motif have non-uniform secondary structures. Thus, the channel architecture evolves as a highly distorted 4-fold pore symmetry.