Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone

Abstract

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

Brugada syndrome trafficking–defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels

“…Interestingly, when a Nav1.5 mutant produces a DNE on Nav1.5, this does not necessarily imply that it concomitantly produces a DNE on Kir2.1/2.2 channels (p.D1690N) and vice versa (p.D1816VfsX7). This reinforces the contention that the biology of Nav1.5-Kir2.1 complexes differs somehow from that of each channel alone, even though the anterograde and retrograde trafficking routes of the complexes are similar to those of Nav1.5 channels alone (7).…”

“…Therefore, the results would indicate that, at least at some stages (biosynthesis and/or forward and retrograde trafficking), Nav1.5 and Kir2.1 proteins do interact, forming complexes. Indeed, previous results from our group demonstrated that at least a pool of Nav1.5 and Kir2.1 channels form complexes early after their synthesis at the ER (8) and that these complexes, whose cellular biology is regulated differently than that of Nav1.5 and Kir2.1 channels alone (7), exhibit preferential forward trafficking toward the membrane (7,8). Interestingly, when a Nav1.5 mutant produces a DNE on Nav1.5, this does not necessarily imply that it concomitantly produces a DNE on Kir2.1/2.2 channels (p.D1690N) and vice versa (p.D1816VfsX7).…”

“…In the 1950s Weidmann (4) established that the encompassed voltage-dependent activity of I Na and I K1 is a major determinant of cardiac excitability and conduction velocity. Furthermore, we demonstrated that the functional expression of Nav1.5 and Kir2.1/Kir2.2 channels is reciprocally regulated (5)(6)(7)(8), a mechanism that probably strengthens control of ventricular excitability.…”

Pharmacological Profile of the Sodium Current in Human Stem Cell-Derived Cardiomyocytes Compares to Heterologous Nav1.5+β1 Model

“…However, β1 is not the only interacting subunit and other auxiliary subunits of Nav1.5 (other β subunits and scaffolding proteins) are probably expressed in hSC-CM (Abriel, 2010). Furthermore, even other channels, such as the Kir2.1 K + channel, can form complexes with Nav1.5 (Willis et al, 2015;Utrilla et al, 2017;Ponce-Balbuena et al, 2018). As these subunits and interactions can modulate the response of Nav1.5, the expression of them in hSC-CMs most likely explains the observed difference in I Na kinetics between hSC-CMs and heterologous expressed Nav1.5+β1 (Figure 1).…”

Altering integrin engagement regulates membrane localization of Kir2.1 channels

“…Since previous studies have shown that Na v 1.5 and K ir 2.1 have a parallelism in expression (Milstein et al, 2012;Utrilla et al, 2017; Ponce-Balbuena et al, 2018), we next explored whether the cell shape and size affect membrane localization and activity of Na v 1.5 channels by immunostaining and electrophysiological analysis, respectively. We found that, unlike K ir 2.1, Na v 1.5 exhibited no preferential localization in the corners versus edges of star-shaped Ex293 cells (Fig.…”