Calmodulin binds FGF12A to regulate Nav1.5 channel inactivation.

Abstract

Activation and inactivation gating of the Nav1.5 voltage-gated ion channel is responsible for the rapid depolarization of the cardiac action potential (AP) and regulates its duration. Altered inactivation kinetics can lead to AP prolongation and potentiate arrhythmia. Th Nav1.5 C-terminal domain (CTD) is known to modulate inactivation. In addition, there are multiple accessory proteins that bind the CTD are CaM and FGF12A. CaM has been linked to a possible Ca2+ modulation of the Nav1.5 as Ca2+ ions typically do not interact directly with the channel. However, the combined interaction on channel inactivation between CaM and FGF12A has also not been determined. We employed the use of cut-open voltage clamping to record the current traces. FRET imaging was used to assess binding affinity between FGF12A and CaM with apparent KD values. The addition of FGF12A to the CTD induces shifts in the activation's voltage dependence (+10.0 ±1.9 SEM mV, p<0.05) and removing CaM with the IQ/AA mutation on Nav1.5 leads to a depolarizing shift in inactivation (+19.5 ±2.1 SEM mV, p<0.01). We have shown that the addition of FGF12A to a channel lacking CaM (IQ/AA) will have a depolarizing shift in the steady state inactivation (+15.5 ±2.6 SEM mV, p<0.01). Furthermore, we have shown that a binding domain for CaM on the FGF12A protein itself requires the presence of Ca2+ ions to associate by measuring increase in the apparent KD value determined through FRET imaging. By removing this CaM binding domain from FGF12A we see a reduction in the recovery from inactivation and a hyperpolarizing shift in the voltage dependence of inactivaiton (−10.0 ±2.8 mV SEM, p<0.0005). The shift in inactivation based upon the presence of CaM could indicate an interaction between Ca2+ ions and the Nav1.5 channel.