Gating control of the cardiac sodium channel Nav1.5 by its β3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain

Abstract

The auxiliary β3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some β3 mutations predispose individuals to cardiac arrhythmias. The β3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the β3 transmembrane region and its potential for functionally synergizing with the β3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT β3-subunit and in a β3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed β3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT β3-subunit. Removal of the β3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the β3-Glu-176 residue and the β3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.

Computational Investigation of Voltage-Gated Sodium Channel β3 Subunit Dynamics

“…Additionally, when the helices of each β3 chain are in close proximity, the conserved E176 residue appears to be orientated away from the trimer center and preferentially interacts with the POPC membrane (data not shown). These observations are also supported by recent experimental work examining the role of E176 in β3 subunits, and that also concluded that oligomerization was dependent on the extracellular domain but not E176 (Salvage et al, 2019).…”

“…This glutamate is highly conserved and is found in both β1 and β3 sequences. Given its unusual position, it has been argued that it is likely to have functional significance and indeed has been investigated within β1 (McCormick et al, 1999) and β3 (Namadurai et al, 2014;Salvage et al, 2019). It also seems that for a full-length model based upon the trimeric β3 crystal structure of the ECD to be adopted in the context of a lipid bilayer system, the TMD helices of our model must change their tilt with respect to the membrane normal.…”

Cell-Adhesion Properties of β-Subunits in the Regulation of Cardiomyocyte Sodium Channels

“…We therefore suggest that unlike β1, the β3-subunit may not play a major role in stabilising the perinexal space by trans -mediated cell-adhesion. On the other hand, when expressed alone in HEK293 cells, the β3-subunits bind homophilically in cis , using their Ig domains [ 19 , 20 , 33 ]. Super-resolution imaging experiments show that when co-expressed with Nav1.5, the β3-subunit affects the relative geometry between Nav1.5 channel α-subunits, possibly by promoting particular orientations of individual Nav1.5 α-subunit dimers within larger clusters [ 20 ].…”

“…They also shift the voltage ranges over which Nav channel steady-state activation and/or inactivation occur, and in some cases enhance the rates of inactivation and recovery from inactivation [ 18 ]. As an illustrative example, the β3-subunit shifts the V ½ for inactivation of Nav1.5 in a depolarising direction: i.e., the voltage at which half the channels are inactivated is displaced to a more positive value compared to the α-subunit alone ( Figure 3 D) [ 19 , 20 , 21 , 22 ]. For a cardiomyocyte with a resting potential of about -90 mV [ 2 ], this would act to increase the fraction of functional Nav1.5 channels available in the membrane [ 17 ].…”

“…Based on biochemical and electrophysiological data, it is probable that the β3-subunit transmembrane alpha-helix also binds to Nav1.5 DIII voltage sensing domain [ 19 , 20 , 21 ]. Yet, there is evidence that it may bind closer to Nav1.5 DIII helix S3 rather than to the binding site for the β1 transmembrane region on the DIII helix S2 [ 19 , 21 ]. If so, then a given Nav1.5 α-subunit may be able to bind simultaneously to β1 and β3-subunits and there is indeed electrophysiological evidence to support this idea [ 21 , 23 ].…”