The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Na_v1.5

Abstract

Background:14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial.Aims:In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5.Methods and Results:Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded byYWHAH) co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The amplitude of Nav1.5-mediated sodium peak current density significantly increased upon co-expression with difopein.Conclusions:Our findings illustrate that the direct implication of 14-3-3 in regulating Nav1.5 is not evident in the heterologous expression system used here. Potentiation of Nav1.5-mediated current by difopein could be explained by the inhibition of 14-3-3/ligand interactions with other partner proteins of the channel complex (e.g., PKA, PKC, CaMKII) and should be further investigated.

A detailed analysis of single‐channel Nav1.5 recordings does not reveal any cooperative gating

“…Although we did not observe functional channelchannel interactions, our results do not exclude the idea that Na + channels can form dimers or multimers (biochemical interaction) as reported in previous studies (Clatot et al, 2017;Iamshanova et al, 2022). Our results also do not exclude the possibility that channels start interacting under conditions of cellular stress.…”

Exploring the Oligomerization of Nav1.5 and Its Implication for the Dominant-Negative Effect

“…10,13 Further confirming the dimerization of heterologous Na v 1.5 occurs in living cells, our group revealed with protein complementation assays that N-N-and C-C-termini arrangement of Na v 1.5 α-subunits was more common than N-C-and C-N-termini arrangement. 17 These results revealed a preference of α-subunits orientation within the dimer. 17 In line with these results, interacting C-termini were reported for the asymmetric cis-dimerization of Na v 1.5 when using Glu1773-Arg1929 peptides (Fig.…”

“…17 These results revealed a preference of α-subunits orientation within the dimer. 17 In line with these results, interacting C-termini were reported for the asymmetric cis-dimerization of Na v 1.5 when using Glu1773-Arg1929 peptides (Fig. 1-2).…”

“…10,16 Direct α-α interaction sites In many studies, co-immunoprecipitation analyses between differently tagged fulllength and truncated Na v 1.5 proteins revealed the channels interact in heterologous expression systems. 9,13,[16][17][18] Analysis of GFP-Na v 1.5 photobleaching steps indicated that the major proportion of α-subunits were monomers (~50%) and dimers (~50%), with the small minority (<5%) being represented by the higher order of oligomers. 10 In addition, Na v 1.5-specific dimers (~460 kDa) were detected in the protein lysates by immunoblotting technique after performing native acrylamide gel electrophoresis and disuccinimidyl suberate cross-linking experiments.…”

“…49 Furthermore, 14-3-3η co-immunoprecipitated with Na v 1.5 in heterologous expression system, while 14-3-3 proteins were pulled down with L1 of Na v 1.5 and co-immunoprecipitated with pan-Na v antibody from mouse heart lysates. 17,49,50 Using yeast two-hybrid assay, Allouis et al, revealed the association between L1 of Na v 1.5 with 14-3-3η and to a lower extent with 14-3-3θ and 14-3-3ζ. 49 According to their findings, the specific interaction site between 14-3-3η and L1 of Na v 1.5 was restricted to Glu417-Ala467, while His445-Glu488, Pro468-Asn507, Arg504-Gln573, Ser560-Leu633 and Glu610-Lys711 fragments of Na v 1.5 did not associate with 14-3-3η (Fig.…”