Salvage SC et al. (2019), Gating control of the cardiac sodium channel Na...

XB-ART-57111

J Biol Chem 2019 Dec 20;29451:19752-19763. doi: 10.1074/jbc.RA119.010283.

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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 SC , Zhu W , Habib ZF , Hwang SS , Irons JR , Huang CLH , Silva JR , Jackson AP .

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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 V ½ 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.

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Species referenced: Xenopus
Genes referenced: ecd nav1

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	Figure 1. Sequence analysis of β3-structures. A, secondary structure prediction of the β3-subunit transmembrane region and its sequence alignment among a wide range of vertebrate species. Residue numbers refer to the human sequence. The predicted transmembrane region is colored orange, and the Glu-176 residue is highlighted. Residues fully conserved between species are indicated with asterisks. B, space-filling model of the β3-subunit with the fully conserved residues of the transmembrane region shown in magenta. Note that the helix face containing the Glu-176 residue is fully conserved. Analysis and modeling were as described under “Experimental procedures.” C, cartoon summary of the WT and mutant β3-subunit constructs used in this work and referred to in the text.
	Figure 2. β3 homooligomerization and Nav1.5α–β3 interaction. A, Western blots (WB) of cell lysates singly transfected with WT-β3-EGFP or mutant subunits, cross-linked with BS3. The monomeric full-length β-subunits tagged with EGFP were run at ∼58–75 kDa. The multiple bands are indicative of variations in glycosylation patterns. With BS3, dimeric and trimeric forms appear at ∼120–130 and 180–200 kDa, respectively. B, co-IP of cell lysates co-transfected with Nav1.5 HA- and EGFP-tagged β3-subunits or control EGFP alone. The samples were immunoprecipitated (IP) with a monoclonal anti-HA. (Nav1.5) antibody coupled to protein G–agarose beads. The bound and flow-through (supernatant) fractions were separated on SDS-PAGE and blotted for HA and EGFP. β3-EGFP with and without the ECD and E176K mutation were pulled down with the Nav1.5 HA. The EGFP is only present in the unbound, flow-through fraction.
	Figure 3. Nav1.5 steady-state activation properties with and without WT or mutant β3. A, representative whole-cell sodium current recordings from HEK293F-Nav1.5 cells transfected with WT and mutant β3-subunits in response to a steady-state activation protocol (inset). B, I/V Boltzmann curves (described under “Experimental procedures”) of Nav1.5 currents normalized to cell capacitance. C, histograms showing the Nav1.5 peak current densities (INa) with and without WT and mutant β3-subunits. D, channel conductance as a function of voltage. Curves are fit to a Boltzmann function; V½ and k were both unaffected by the presence of the β3-subunit or any of the mutants. For B–D, the data are means ± S.D. (n ≥ 6; see Table 1 for individual groups), and statistical significance was tested with one-way ANOVA. All parameters (peak INa, V½, and k) showed no statistically significant variation (p > 0.2). See Table 1 for individual values.
	Figure 4. Effects of the β3-subunit on Nav1.5 steady-state inactivation are mediated through the ECD. A, representative whole-cell Na+ currents in response to a steady-state inactivation protocol (inset). B, peak INa from each sweep normalized to the maximum peak INa (INa/INa.max) of all sweeps and plotted as a function of the pre-pulse voltage step. The data are means ± S.D. (n ≥ 10, see Table 1 for individual values) and are separated by Nav1.5 + EGFP, WT-β3-EGFP, and β3-E176K-EGFP in the left panel and Nav1.5 + EGFP, ΔECD-β3-EGFP, and ΔECD-β3-E176K-EGFP in the right panel. The Nav1.5 + EGFP is the same in both graphs; the separation is for clarity. The curves are fit to Boltzmann functions (see “Experimental procedures”). The statistical significance of the V½ and k values produced were determined using one-way ANOVA (both V½ and k; p < 0.01) followed by a Sidak's multiple comparison post hoc test (all conditions were compared against Nav1.5 + EGFP and Nav1.5 + β3-EGFP). V½ of Nav1.5 steady-state inactivation is positively shifted by the full-length β3 and the β3 with the single transmembrane E176K point mutation (Nav1.5 + EGFP versus WT-β3, p = 0.0026; and Nav1.5 + EGFP versus β3-E176K-EGFP, p = 0.0263). Removal of the Ig-like ECD abolishes these shifts (Nav1.5 + EGFP versus β3-ΔECD-EGFP, p = 0.865; and Nav1.5 + EGFP versus β3-ΔECD-E176K-EGFP, p = 0.99). See Table 1 for all comparisons.
	Figure 5. Acceleration of Nav1.5 recovery from inactivation by β3 is abolished with loss of the ECD or the transmembrane glutamic acid. Recovery from inactivation is expressed as the fraction of current produced by a second pulse over time following an identical pre-pulse (see “Experimental procedures”). The data are means ± S.D. (n ≥ 7, see Table 2) fit to double exponential functions, and the parameters (k, τ, and t½ for both the fast and slow components) are compared with one-way ANOVA (p ≤ 0.002 for all) followed by a Sidak's multiple comparison post hoc test (all conditions were compared against Nav1.5 + EGFP and Nav1.5 + β3-EGFP). Nav1.5 + EGFP, WT-β3-EGFP, and β3-E176K-EGFP are in the left panel, and Nav1.5 + EGFP, ΔECD-β3-EGFP, and β3-ΔECD-E176K-EGFP are in the right panel. The Nav1.5 + EGFP is the same in both graphs; the separation is for clarity. Nav1.5 recovery from inactivation was accelerated by the presence of the WT full-length β3-subunit (Nav1.5 + EGFP versus WT-β3-EGFP, p < 0.001 for all fast and slow components). This effect was lost with the full-length β3-E176K mutation (Nav1.5 + EGFP versus β3-E176K-EGFP, p > 0.05) and loss of the ECD (Nav1.5 + EGFP versus β3-ΔECD). See Table 2 for full details.
	Figure 6. Effects of the β3-subunits on Nav1.5 VSD movements as detected by VCF. A, voltage dependence of fluorescence changes in DIII and DIV of Nav1.5. All curves are fit with a Boltzmann function, and the corresponding data points are means ± S.D. (n ≤ 3, see Table 3) compared by one-way ANOVA (all parameters p ≤ 0.0005) and followed by a Sidak's multiple comparison post hoc test (all conditions were compared against Nav1.5 + EGFP and Nav1.5 + β3-EGFP; see Table 3 for full results). Upper left panel, normalized change in DIII fluorescence plotted (means ± S.D.) as a function of test potential (F–V curve) for Nav1.5 α alone and with WT-β3 or β3-E176K. WT-β3 induces a depolarizing shift in the F–V curve compared with Nav1.5 alone (Nav1.5 versus Nav1.5 + β3-EGFP; p = 0.0002), an effect lost in the presence of the E176K mutation (Nav1.5 + β3-EGFP versus Nav1.5 + β3-E176K-EGFP; p < 0.0001). Adjacent insets show corresponding representative fluorescence signals and test protocol. Upper right panel, DIV F–V curve for α alone and with WT-β3 or β3-E176K. The presence of either WT-β3 or β3-E176K induces a depolarizing rightward shift in the initial activation of DIV, but the movement is accelerated compared with Nav1.5 alone. Adjacent insets show corresponding representative VCF traces and test protocol. Lower left panel, DIII F–V curve for α alone or with β3-ΔECD or β3-ΔECD-E176K. The loss of the ECD results in a hyperpolarizing leftward shift (Nav1.5 + β3-EGFP versus Nav1.5 + β3-ΔECD, p < 0.0001; and Nav1.5 + β3-EGFP versus Nav1.5 + β3-ΔECD-E176K-EGFP, p < 0.0001). Adjacent insets show the corresponding representative fluorescence signals, where the movement of the DIII sensor is noticeably attenuated by the presence of either β3-subunits lacking the ECD. Lower right panel, DIV F–V curve for α alone or with β3-ΔECD or β3-ΔECD-E176K. β3-ΔECD and β3-ΔECD-E176K co-expression result in differing degrees of rightward depolarizing shift of the curve. Adjacent insets show the corresponding representative fluorescence signals, where the movement of the DIV sensor like the DIII sensor is noticeably attenuated. B, histograms of the mean (± S.D., n ≥ 3) V½ (left panel) and k (right panel) values for DIII and DIV from the Boltzmann curve fits of the data in A. Table 3 has the full numerical values and statistical comparisons.

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