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Gating control of the cardiac sodium channel Nav1.5 by its ?3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

ABSTRACT: 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 ?3-subunit uses its transmembrane ?-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 ?3 transmembrane region and its potential for functionally synergizing with the ?3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT ?3-subunit and in a ?3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed ?3-dependent depolarizing shift of V ½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT ?3-subunit. Removal of the ?3-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 ?3-Glu-176 residue and the ?3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.

SUBMITTER: Salvage SC

PROVIDER: S-EPMC6926464 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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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.

Salvage Samantha C SC Zhu Wandi W Habib Zaki F ZF Hwang Soyon S SS Irons Jennifer R JR Huang Christopher L H CLH Silva Jonathan R JR Jackson Antony P AP

The Journal of biological chemistry 20191028 51

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- ...[more]

PMID: 31659116

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Project description:Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated β-subunits. The β3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length β3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the β3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the β3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3-subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the β3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the β3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the β3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

Dataset Information

Gating control of the cardiac sodium channel Nav1.5 by its ?3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

Publications

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.

Similar Datasets

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