Title

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

Document Type

Article

Publication Date

1-1-1993

Abstract

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 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation‐selective channels, but with distinct characteristics: the single‐channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by μM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 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. Copyright © 1993 The Protein Society

Publication Source (Journal or Book title)

Protein Science

First Page

1918

Last Page

1930

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