Abstract Body: Introduction
Voltage-gated calcium channels (Ca_v1.2, CACNA1C) activate during the plateau phase of the cardiac action potential (AP). Negative feedback mechanisms that control the channel inactivation are located in the cytosolic C-terminus of Ca_v1.2.
Despite efforts, the structure of the C-terminus of Ca_v1.2 has not yet been fully resolved.
Hypothesis
We characterised a variant of uncertain significance located within the EF-hand domain and hypothesized that L1521I-Ca_v1.2, reported in a cardiology patient, altered calcium-dependent inactivation (CDI).
Aims
To clarify biophysical properties of L1521I-Ca_v1.2 given its location within the EF-hand domain.
Methods
The patch-clamp technique was used to record currents and biophysical properties of wt-Ca_v1.2 and L1521I-Ca_v1.2 channels expressed in doxycycline-inducible Flp-In HEK293 cells. Prediction of the functional effects of L1521I-Ca_v1.2 was performed using the PolyPhen-2 bioinformatic tool. Models of the mutant EF-hand domain were prepared using COOT and geometry optimization.

Results
Amplitude of the L1521I-Ca_v1.2 channel current did not differ from wt-Ca_v1.2. However, slope (L1521I: 9.9±0.3 mV n=38 vs wt: 8.1±0.33 mV n=53, p<0.001) and V_1/2 (L1521I: 13.2±1.6 mV n=38 vs wt: 5.3±1.6 mV n=53, p<0.001) of the channel fractional activation were altered.
V_1/2 of steady-state activation was right-shifted in L1521I-Ca_v1.2 channels (15.3±2.0 mV n=11, vs wt: 8.6±1.7 mV n=19, p<0.05), and window current increased (4.5±0.6 a.u. n=11 vs wt: 2.7±0.3 a.u., n=19 p<0.05).
L1521I-Ca_v1.2 delayed CDI and this difference was statistically significant 225 ms after channel activation.
Conclusions
Our electrophysiological data suggest that L1521I-Ca_v1.2 is a gain-of-function mutation. L1521I delayed CDI of Ca_v1.2, right-shifted V_1/2 of steady-state activation and augmented window current.
L1521 is part of a hydrophobic core and is completely buried. Although in our COOT and geometry optimization model, L1521I mutation is predicted to cause only small perturbations in the Ca_v1.2 structure, we speculate that allosterically it may be sufficient to affect interaction between EF-hand and III-IV linker. Consistent with our in silico modelling, these alterations may be sufficient to prolong the AP and QT interval on ECG.