14-3-3 is a regulator of the cardiac voltage-gated sodium channel Nav1.5.
The voltage-sensitive Na(+) channel Na(v)1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3eta, interacting with the Na(v)1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Na(v)1.5 both in COS-7 cells expressing recombinant Na(v)1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Na(v)1.5 colocalized at the intercalated discs. We tested the functional link between Na(v)1.5 and 14-3-3eta using the whole-cell patch-clamp configuration. Coexpressing Na(v)1.5, the beta1 subunit and 14-3-3eta induced a negative shift in the inactivation curve of the Na(+) current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Na(v)1.5 cytoplasmic I interdomain interacting with 14-3-3eta. Reversal was also obtained with the dominant negative R56,60A 14-3-3eta mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na(+) channel acting as a cofactor for the regulation of the cardiac Na(+) current.
Allouis
Marie
M
Inserm UMR533, Université de Nantes, Nantes Atlantique Universités, l'Institut du thorax, Faculté de Médecine, France.
Le Bouffant
Françoise
F
Wilders
Ronald
R
Péroz
David
D
Schott
Jean-Jacques
JJ
Noireaud
Jacques
J
Le Marec
Hervé
H
Mérot
Jean
J
Escande
Denis
D
Baró
Isabelle
I
D048948:14-3-3 Proteins / Q000737:chemistry / Q000502:physiology*; D000200:Action Potentials / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D019281:Dimerization; D004553:Electric Conductivity; D004594:Electrophysiology; D006321:Heart / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008955:Models, Cardiovascular; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms / Q000502:physiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D048948:14-3-3 Proteins; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; D011994:Recombinant Proteins; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels