Inter-Regulation of Kv4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

Abstract

Background: Genetic variants in voltage-gated sodium channels (Na_v) encoded by SCNXA genes, responsible for I_Na, and K_v4.3 channels encoded by KCND3, responsible for the transient outward current (I_to), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that K_v4.3 and Na_v variants regulate each other's function, thus modulating I_Na/I_to balance in cardiomyocytes and I_Na/I_(A) balance in neurons.

Methods: Bicistronic and other constructs were used to express WT or variant Na_v1.5 and K_v4.3 channels in HEK293 cells. I_Na and I_to were recorded.

Results: SCN5A variants associated with BrS reduced I_Na, but increased I_to. Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of I_to, significantly reduced I_Na, whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of I_to significantly increased I_Na. Auxiliary subunits Na_vβ1, MiRP3 and KChIP2 also modulated I_Na/I_to balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase K_v4.3 cell-surface expression.

Conclusion: Na_v and K_v4.3 channels modulate each other's function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.

Keywords: Brugada syndrome; KCND3; Kv4.3; Nav1.1; Nav1.5; SCN1A; SCN5A; arrhythmia; channelopathies; spinocerebellar ataxia.

Figure 1

The presence of Na_v1.5 variants affects outward current (I_to). (A) I_to current–voltage relationships recorded from HEK293 cells co-expressing K_v4.3-short (pGFP-IRES-KCND3-Short) channels and either WT, R878C, G1743R, or E555X-Na_v1.5 channels (pcDNA3.1-GFP-SCN5A). (B): Representative current traces of I_Na in the prepulse followed by I_to. Inset shows the voltage protocol employed. The presence of Na_v1.5 variants significantly affect I_to compared to WT, at + 20 mV, *** p < 0.001 for the three variants (In pA/pF; WT = 87.14 ± 8.2, R878C = 243.3 ± 57.55, G1743R = 54.8 ± 12.9, E555X = 138 ± 16.8) (C): I_to steady-state inactivation. Note: G1743R-Na_v1.5 significantly shifts the steady-state inactivation V_1/2 of I_to compared to WT-Na_v1.5, *** p < 0.001. n represents the number of recorded cells. In panel A, n = 50 WT cells correspond to total of WT cells patched against each variant.

Figure 2

K_V4.3 variants affect I_Na. (A): Representative traces of I_to and I_Na measured in HEK293 cells co-expressing the bicistronic construct Na_v1.5/K_v4.3-WT, -Δ227F or -L450F (pKCND3-Short-poliovirus-SCN5A) with Na_vβ1/GFP reporter gene (pGFP-IRES-SCN1B). (B): Normalized current–voltage relationships, the trafficking-efficient L450F leads to an increase of I_to and a significant decrease in I_Na at potentials positive to −35 mV p = 0.039 (p < 0.001 at −20 mV), whereas the trafficking-deficient Δ227F-K_v4.3 leads to a decrease of I_to but a significant increase in I_Na at potentials positive to −45 mV p = 0.018 (p < 0.001 at −20 mV). (C): Steady state inactivation of I_Na. K_v4.3 variants did not affect I_Na steady-state inactivation. (D): Normalized current–voltage relationship in HEK293 cells stably expressing Na_v1.1, Na_vβ1 and Na_vβ2 and transfected with K_v4.3-S WT vs mutants (pGFP-IRES-KCND3-short). The trafficking-efficient L450F leads to an increase of I_to but a significant decrease in I_Na at potentials positive to −30 mV p = 0.04 (p < 0.001 at −5 mV), whereas the trafficking-deficient Δ227F-K_v4.3 leads to a decrease of I_to but a significant increase in I_Na at potentials positive to −20 mV p = 0.05 (p < 0.001 at −5 mV). Note: n represents the number of cells recorded.

Figure 3

Na_vβ1 and MiRP3 decrease I_to and increase I_Na. HEK293 cells were transfected with the bicistronic construct Na_v1.5/K_v4.3 (pKCND3-Short-poliovirus-SCN5A), with or without Na_Vβ1 (pGFP-IRES-SCN1B vs pGFP) or MiRP3 (pRFP-IRES-KCNE4 vs pRFP) (A,B), or with Na_v1.5 (pcDNA3.1-GFP-SCN5A)(C), or K_v4.3 (pGFP-IRES-KCND3-Short)(D) with MiRP3. (A): Current–voltage relationship measured in HEK293 cells co-expressing the bicistronic construct Na_v1.5/K_v4.3 (pKCND3-Short-poliovirus-SCN5A), with or without Na_Vβ1 (pGFP-IRES-SCN1B vs pGFP) showing significant I_Na increase in presence of Na_vβ1 at potentials positive to −45 mV p < 0.001 (p < 0.001 at −20 mV) and I_to decrease in presence of Na_vβ1 at potentials positive to −5 mV p = 0.049 (p < 0.001 at +40 mV) (B): I_Na increases significantly in presence of MiRP3 at potentials positive to −45 mV p < 0.001 (p < 0.001 at −40 mV) while I_to decreases significantly at potentials positive to −10 mV p = 0.039 (p < 0.001 at +40 mV). (C): In absence of K_v4.3, MiRP3 has no effect on I_Na. (D): In absence of Na_v1.5, MiRP3 decrease I_to at potentials positive to −20 mV p < 0.001 (p < 0.001 at +40 mV) Note: β-subunits that decrease I_to lead to a significant increase of I_Na only if both channels, Na_v1.5 and K_v4.3, are present. n represents the number of cells recorded.

Figure 4

KChIP2 known to increase I_to decrease I_Na. HEK293 cells were transfected with either the bicistronic construct Na_v1.5/K_v4.3 (pKCND3-Short-poliovirus-SCN5A) (A, C), or with Na_v1.5 (pGFP-SCN5A) (B), with or without KChIP2 (pGFP-KCNIP2 vs pGFP), Na_vβ1 (pGFP-IRES-SCN1B vs pGFP), or MiRP3 (pRFP-IRES-KCNE4 vs pRFP). (A): Current–voltage relationships show that I_Na is significantly decreased in the presence of KChIP2 at potentials positive to −45 mV p = 0.024 (p = 0.002 at −40 mV), whereas I_to is significantly increased at potentials positive to −40 mV p = 0.009 (p < 0.001 at +45 mV). (B): In absence of K_v4.3, KChIP2 has no effect on I_Na. n represents the number of cells recorded.

Figure 5

Na_v1.5 and K_v4.3 interact with each other. (A): Co-immunoprecipitation between Na_v1.5 and K_v4.3. HEK293 cells were transfected with WT or truncated Na_v1.5 constructs and K_v4.3 (pcDNA3.1-GFP-SCN5A and pCMV-KCND3-Long-Flag) as indicated above the lanes. The total cell lysates were immunoprecipitated with an anti-Flag antibody, specific to K_v4.3-Flag, cross-linked to beads. The blots were hybridized with an anti-Na_v1.5 antibody (top gels: Blot Ab: Na_v1.5) or an anti-Flag antibody (bottom gels: Blot Ab: Flag). The left side corresponds to the total cell lysates of transfected cells before IP. The right side (IP with Flag Ab) corresponds to the elution fractions from beads. The negative control (center of panel A), consisting in an anti-Flag immunoprecipitation in lysates of cells expressing only GFP-Na_v1.5 channels, clearly excluded any non-specific interaction between Na_v1.5 and K_v4.3 channels. The results demonstrated an interaction between K_v4.3 and Na_v1.5 (n = 7). (B): Duolink between GFP-Na_v1.5 and K_v4.3-Flag. The top line corresponds to cells co-expressing GFP alone (pGFP) with K_v4.3-Flag while the bottom line cells co-expressing GFP-Na_v1.5 with K_v4.3-Flag. Only cells expressing GFP-Na_v1.5 and K_v4.3-Flag display red positive signals indicating a close proximity between the two channels. Note: Close proximity of the two channels can be observed within intracellular compartments. (C): Cell surface biotinylation of K_v4.3 in presence of WT, R878C or G1743R GFP-Na_v1.5 channels. TP: Total Protein, IC: Intracellular, S: Surface. Note that values of S abundance were not directly quantitated from blots, but calculated as detailed in the method section. The presence of R878C significantly increases the cell surface expression of K_v4.3, * p = 0.033 consistent with the observed increase of I_to. Note: In cells co-expressing Na_v1.5 and K_v4.3, the two channels were co-immunoprecipitated.

Figure 6

Schematic of the influence of β-subunits of the two channels on the I_Na/I_to balance. Left panel represents raw traces of I_Na/I_to displaying a larger I_Na and smaller I_to as opposed to a smaller I_Na and larger I_to that could potentially lead to the expression of BrS. The right schematic shows a schematic of the influence of the β-subunits of the two channels on the I_Na/I_to balance.