Acute PKA modulation of the cardiac sodium current is mediated by 14-3-3.

Abstract

Nav1.5 is an essential component of the cardiac action potential. Dysregulation of Nav1.5 contributes to cardiac arrhythmias and Nav1.5 mutations have been linked to lethal congenital cardiac diseases. However, myriad aspects of Nav1.5 function and regulation, mainly as it relates to associated regulatory proteins, remain to be elucidated. PKA or β-adrenergic stimulation is a regulator of cardiac function, and could modulate Nav1.5 function under both healthy and diseased conditions. 14-3-3 is a Nav1.5 interacting protein. Mass spectrometry studies revealed proximity between Nav1.5 PKA phosphorylation sites and the 14-3-3 binding site. Importantly, the 14-3-3 binding site on Nav1.5 was suggested to be phosphorylated by PKA. This led us to hypothesize that PKA could acutely modulate Nav1.5 through 14-3-3. We studied acute modulation by PKA of Nav1.5 using high-throughput automated patch clamping. Our data showed that acute PKA modifications could significantly change the sodium current. Interestingly, inhibition of 14-3-3 abolished the acute PKA effects. Serine 460 is part of the putative 14-3-3 binding motif and mutating Serine 460 to alanine (S460A) behaves like 14-3-3 inhibition and uncouples sodium currents. We therefore tested the effect of PKA modulation on Nav1.5-S460A and found that similarly to 14-3-3 inhibition, PKA had no effect on Nav1.5-S460A currents. To confirm the specific involvement of S460 and 14-3-3 in PKA modulation of Nav1.5, we mutated other phosphorylation sites found in the DI-DII linker. We found that except for S460A, all the other phospho-inhibiting mutations responded to PKA modulation. Our study demonstrated that PKA or β-adrenergic stimulation can acutely modulate Nav1.5 through protein 14-3-3 and Nav1.5-S460 appears to be central to this modulation. We conclude that 14-3-3 does not only regulate the Nav1.5 function and coupling, but also plays a key role in the PKA modulation.