Linker flexibility of IVS3-S4 loops modulates voltage-dependent activation of L-type Ca2+ channels

Extracellular S3-S4 linkers of domain IV (IVS3-S4) of L-type Ca2+ channels (Ca(V)1) 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 Ca(V)1 channels. In this study, we systematically compared IVS3-S4 variants from Ca(V)1.1-1.4, 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-1/2) or activation time constant (tau) are positively correlated with the numbers of flexible glycine residues within the linker. Moreover, antibodies that reduce IVS3-S4 flexibility negatively shifted V-1/2, emerging as a new category of Ca(V)1 enhancers. In summary, our results suggest that the flexibility or rigidity of IVS3-S4 linker underlies its modulations on Ca(V)1 activation (V-1/2 and tau), paving the way to dissect the core mechanisms and to develop innovative perturbations pertaining to voltage-sensing S4 and its vicinities.