Key result
Disrupting the Cav3.2 helix-loop-helix gating brake shifts activation curves and accelerates channel kinetics.
Mutations in the gating brake of the I-II loop of Cav3.2 channels disrupt its structural integrity, preventing the channels from remaining closed at resting membrane potentials.
May promote Cav3.2 opening at rest in disease models; leaves open clinical relevance without human validation.
Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, 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 had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.
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Arias‐Olguín et al. (2008) studied Childhood absence epilepsy. Mutagenesis of the gating brake in the I-II loop of Cav3.2 channels vs. Control (wild-type channels) was evaluated on Channel gating (steady state activation and inactivation curves, channel kinetics). Mutations disrupting the helix-loop-helix structure of the gating brake in the I-II loop of Cav3.2 channels shifted activation and inactivation curves and accelerated channel kinetics.
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