Characterization of the gating brake in the I-II loop of CaV3 T-type calcium channels

Our interest was drawn to the I-II loop of Ca(V)3 channels for two reasons: (1) transfer of the I-II loop from a high voltage-activated channel (Ca(V)2.2) to a low voltage-activated channel (Ca(V)3.1) unexpectedly produced an ultra-low voltage activated channel; and (2) sequence variants of the I-II loop found in childhood absence epilepsy patients altered channel gating and increased surface expression of Ca(V)3.2 channels. To determine the roles of this loop we have studied the structure of the loop and the biophysical consequences of altering its structure. Deletions localized the gating brake to the first 62 amino acids after IS6 in all three Ca(V)3 channels, establishing the evolutionary conservation of this region and its function. Circular dichroism was performed on a purified fragment of the I-II loop from Ca(V)3.2 to reveal a high a-helical content. De novo computer modeling predicted the gating brake formed a helix-loop-helix structure. This model was tested by replacing the helical regions with poly-proline-glycine (PGPGPG), which introduces kinks and flexibility. These mutations had profound effects on channel gating, shifting both steady-state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure (poly-alanine, which forms a-helices) had more modest effects. Taken together, we conclude the second helix of the gating brake establishes important contacts with the gating machinery, thereby stabilizing a closed state of T-channels, and that this interaction is disrupted by depolarization, allowing the S6 segments to spread open and Ca2+ ions to flow through.