Linker flexibility of IVS3-S4 loops modulates voltage-dependent activation of L-type Ca channels.

Extracellular S3-S4 linkers of domain IV (IVS3-S4) of L-type Ca channels (Ca1) are subject to alternative splicing, resulting into distinct gating profiles serving for diverse physiological roles. However, it has remained elusive what would be the determining factor of IVS3-S4 effects on Ca1 channels. In this study, we systematically compared IVS3-S4 variants from Ca1.1-1.4, and discover that the flexibility of the linker plays a prominent role in gating characteristics. Chimeric analysis and mutagenesis demonstrated that changes in half activation voltage (V) or activation time constant (τ) are positively correlated with the numbers of flexible glycine residues within the linker. Moreover, antibodies that reduce IVS3-S4 flexibility negatively shifted V, emerging as a new category of Ca1 enhancers. In summary, our results suggest that the flexibility or rigidity of IVS3-S4 linker underlies its modulations on Ca1 activation (V and τ), paving the way to dissect the core mechanisms and to develop innovative perturbations pertaining to voltage-sensing S4 and its vicinities.