Journal: International Journal of Molecular Sciences

Article Title: Inter-Regulation of K v 4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

doi: 10.3390/ijms21145057

Figure Lengend Snippet: The presence of Na v 1.5 variants affects outward current (I to ). ( A ) I to current–voltage relationships recorded from HEK293 cells co-expressing K v 4.3-short (pGFP-IRES-KCND3-Short) channels and either WT, R878C, G1743R, or E555X-Na v 1.5 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 v 1.5 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 v 1.5 significantly shifts the steady-state inactivation V 1/2 of I to compared to WT-Na v 1.5, *** 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.

Article Snippet: After washing the beads 4 times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37 °C for 30 min under agitation, separated on a NuPAGE 7% Tris-Acetate gel (Invitrogen), transferred to a nitrocellulose membrane and incubated with primary antibodies: mouse anti-Flag (1:500, Sigma-Aldrich), rabbit anti-Na v 1.5 antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-transferrin receptor (1:500, Invitrogen) as a loading control.

Techniques: Expressing, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Inter-Regulation of K v 4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

doi: 10.3390/ijms21145057

Figure Lengend Snippet: K V 4.3 variants affect I Na . ( A ): Representative traces of I to and I Na measured in HEK293 cells co-expressing the bicistronic construct Na v 1.5/K v 4.3-WT, -Δ227F or -L450F (pKCND3-Short-poliovirus-SCN5A) with Na v β1/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 v 4.3 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 v 4.3 variants did not affect I Na steady-state inactivation. ( D ): Normalized current–voltage relationship in HEK293 cells stably expressing Na v 1.1, Na v β1 and Na v β2 and transfected with K v 4.3-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 v 4.3 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.

Article Snippet: After washing the beads 4 times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37 °C for 30 min under agitation, separated on a NuPAGE 7% Tris-Acetate gel (Invitrogen), transferred to a nitrocellulose membrane and incubated with primary antibodies: mouse anti-Flag (1:500, Sigma-Aldrich), rabbit anti-Na v 1.5 antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-transferrin receptor (1:500, Invitrogen) as a loading control.

Techniques: Expressing, Construct, Stable Transfection, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Inter-Regulation of K v 4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

doi: 10.3390/ijms21145057

Figure Lengend Snippet: Na v β1 and MiRP3 decrease I to and increase I Na . HEK293 cells were transfected with the bicistronic construct Na v 1.5/K v 4.3 (pKCND3-Short-poliovirus-SCN5A), with or without Na V β1 (pGFP-IRES-SCN1B vs pGFP) or MiRP3 (pRFP-IRES-KCNE4 vs pRFP) ( A , B ), or with Na v 1.5 (pcDNA3.1-GFP-SCN5A)(C), or K v 4.3 (pGFP-IRES-KCND3-Short)(D) with MiRP3. ( A ): Current–voltage relationship measured in HEK293 cells co-expressing the bicistronic construct Na v 1.5/K v 4.3 (pKCND3-Short-poliovirus-SCN5A), with or without Na V β1 (pGFP-IRES-SCN1B vs pGFP) showing significant I Na increase in presence of Na v β1 at potentials positive to −45 mV p < 0.001 ( p < 0.001 at −20 mV) and I to decrease in presence of Na v β1 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 v 4.3, MiRP3 has no effect on I Na . ( D ): In absence of Na v 1.5, 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 v 1.5 and K v 4.3, are present. n represents the number of cells recorded.

Article Snippet: After washing the beads 4 times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37 °C for 30 min under agitation, separated on a NuPAGE 7% Tris-Acetate gel (Invitrogen), transferred to a nitrocellulose membrane and incubated with primary antibodies: mouse anti-Flag (1:500, Sigma-Aldrich), rabbit anti-Na v 1.5 antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-transferrin receptor (1:500, Invitrogen) as a loading control.

Techniques: Transfection, Construct, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inter-Regulation of K v 4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

doi: 10.3390/ijms21145057

Figure Lengend Snippet: KChIP2 known to increase I to decrease I Na . HEK293 cells were transfected with either the bicistronic construct Na v 1.5/K v 4.3 (pKCND3-Short-poliovirus-SCN5A) (A, C), or with Na v 1.5 (pGFP-SCN5A) (B), with or without KChIP2 (pGFP-KCNIP2 vs pGFP), Na v β1 (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 v 4.3, KChIP2 has no effect on I Na . n represents the number of cells recorded.

Article Snippet: After washing the beads 4 times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37 °C for 30 min under agitation, separated on a NuPAGE 7% Tris-Acetate gel (Invitrogen), transferred to a nitrocellulose membrane and incubated with primary antibodies: mouse anti-Flag (1:500, Sigma-Aldrich), rabbit anti-Na v 1.5 antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-transferrin receptor (1:500, Invitrogen) as a loading control.

Techniques: Transfection, Construct

Journal: International Journal of Molecular Sciences

Article Title: Inter-Regulation of K v 4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

doi: 10.3390/ijms21145057

Figure Lengend Snippet: Na v 1.5 and K v 4.3 interact with each other. ( A ): Co-immunoprecipitation between Na v 1.5 and K v 4.3. HEK293 cells were transfected with WT or truncated Na v 1.5 constructs and K v 4.3 (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 v 4.3-Flag, cross-linked to beads. The blots were hybridized with an anti-Na v 1.5 antibody (top gels: Blot Ab: Na v 1.5) 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 v 1.5 channels, clearly excluded any non-specific interaction between Na v 1.5 and K v 4.3 channels. The results demonstrated an interaction between K v 4.3 and Na v 1.5 ( n = 7). ( B ): Duolink between GFP-Na v 1.5 and K v 4.3-Flag. The top line corresponds to cells co-expressing GFP alone (pGFP) with K v 4.3-Flag while the bottom line cells co-expressing GFP-Na v 1.5 with K v 4.3-Flag. Only cells expressing GFP-Na v 1.5 and K v 4.3-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 v 4.3 in presence of WT, R878C or G1743R GFP-Na v 1.5 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 v 4.3, * p = 0.033 consistent with the observed increase of I to . Note: In cells co-expressing Na v 1.5 and K v 4.3, the two channels were co-immunoprecipitated.

Article Snippet: After washing the beads 4 times with PBS-tween 0.02%, proteins were eluted with the Laemmli sample buffer at 37 °C for 30 min under agitation, separated on a NuPAGE 7% Tris-Acetate gel (Invitrogen), transferred to a nitrocellulose membrane and incubated with primary antibodies: mouse anti-Flag (1:500, Sigma-Aldrich), rabbit anti-Na v 1.5 antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-transferrin receptor (1:500, Invitrogen) as a loading control.

Techniques: Immunoprecipitation, Transfection, Construct, Negative Control, Expressing