Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels

To identify sequence‐specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel‐forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage‐gated calcium channel. The amino acid sequence of voltage‐gated, dihydropyridine (DHP)‐sensitive calcium channels suggests the presence in each of four homologous repeats (I–IV) of six segments (S1–S6) predicted to form membrane‐spanning, α ‐helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four‐helix bundle motif, four‐helix bundle proteins representing IVS2 (T 4 CaIVS2) or IVS3 (T 4 CaIVS3) were synthesized. Both proteins form cation‐selective channels, but with distinct characteristics: the single‐channel conductance in 50 mM BaCl 2 is 3 pS and 10 pS. For T 4 CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba 2+ , Ca 2+ , and Sr 2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T 4 CaIVS2 does not saturate up to 150 mM salt. Whereas T 4 CaIVS3 is blocked by μ M Ca 2+ and Cd 2+ , T 4 CaIVS2 is not blocked by divalent cations. Only T 4 CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T 4 CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.