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

Samantha C Salvage,Wandi Zhu,Zaki F Habib,Soyon S Hwang,Jennifer R Irons,Christopher L.H Huang,Jonathan R Silva,Antony P Jackson

doi:10.1074/jbc.ra119.010283

Samantha C Salvage, Wandi Zhu + Show 6 more

Open Access

https://doi.org/10.1074/jbc.ra119.010283

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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.

Highlights

The auxiliary ␤3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ␤3 mutations predispose individuals to cardiac arrhythmias
The Nav channel is composed of a 250-kDa ␣-subunit, which consists of four homologous domains (DI–DIV) containing the voltage-sensing domains (VSDs)5 and the ion-selective pore, in association with one or more auxiliary ␤-subunits that modify the gating behavior of the channel [1]
Recent structural evidence suggests that the DIII and DIV VSDs are implicated in the control of Nav channel inactivation [11, 12]

Summary

Edited by Mike Shipston

The auxiliary ␤3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ␤3 mutations predispose individuals to cardiac arrhythmias. Voltage-clamp fluorometry (VCF) experiments strongly imply that in Nav1.5, the ␤3-subunit influences the gating behavior of both these VSDs [13, 14]. Consistent with this evidence, the ␤3-subunit accelerates Nav1.5 recovery from the inactivated state and produces a shift in the voltage, V1⁄2, of steady-state inactivation [13, 15]. We examine the effects of these mutations on gating behavior and voltage-dependent movements of Nav1.5 DIII and DIV VSDs, using whole-cell patch-clamp electrophysiology and VCF. Our results suggest that the ␤3-subunit transmembrane ␣-helix lies close to the DIII VSD, where the Glu176 residue plays an important role in facilitating recovery from inactivation. These data provide new structural and functional insights into how the ␤3-subunit can modulate both steady-state inactivation and recovery from inactivation

Results

Peak INa

Recovery from inactivation kfast kslow

Discussion

Secondary structure predictions and modeling

Voltage protocols