Molecular basis of NaV1.5 Calcium modulation

Voltage-gated sodium (NaV) channels in the heart are responsible for generating the action potential upstroke that initiates excitation-contraction coupling. The significance of our research lies in the fact that in the heart, even relatively mild perturbations of NaV1.5 due to genetic mutation or pharmacological inhibition, can lead to conduction slowing, cardiac arrhythmias, myopathies, seizures, and sudden cardiac death. Pathophysiologic conditions including ischemia elicit a rise in intracellular free calcium (Cai). We have previously observed that steady-state inactivation properties of Nav1.5 are altered in response to changing Ca2+ and have identified three cytoplasmic regions that are involved: (i) an EF-hand domain that directly binds Ca2+ and (ii) an IQ motif located just downstream from the EF-hand domain. Remarkably, the IQ motif appears to play a dual role, recruiting Calmodulin (CaM) to NaV1.5 and regulating the Ca2+ affinity of the EF-hand domain. Our recent work has extended this complex model for Cai sensing in NaV1.5 to include the cytoplasmic linker between domains 3 and 4 (D3D4 linker), a region known to be involved in inactivation gating. This is highly innovative because the direct effects of Ca2+ on Nav1.5 are not well studied. The primary goal of this proposal is to elucidate the mechanism of action of this complex calcium sensing apparatus at the molecular level. Our central hypothesis is that coupling of the EF-hand domain and CaM through sharing of the IQ motif finely tunes the response of NaV1.5 to Cai, and that action of this complex Cai sensing apparatus through the D3D4 linker leads to modulation of inactivation gating of Nav1.5. Our 3 Aims seek to systematically characterize the structural and functional basis of NaV1.5 Cai sensing. Yeast-Two-Hybrid interactions will determine whether interactions between different components of the Ca2+-sensing apparatus can be disrupted by introducing mutations that are rationally predicted based on our preliminary structural data. We will then use patch-clamp to determine the effects of these mutations on Nav1.5 inactivation. Aim 1 addresses the EF-hand/IQ domain interaction, Aim 2 the IQ motif/CaM interaction, and Aim 3 the coupling of the Cterminal Cai apparatus to D3D4 linker by CaM. This research will a more detailed understanding of the mechanism of the Nav1.5 Cai sensing apparatus and its role in inactivation gating opening up new avenues for developing antiarrhythmic strategies.