The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes.
Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.
Abi-Char
Joëlle
J
INSERM UMR 621, Faculté de Médecine Pierre-Marie Curie, 91 boulevard de l'Hôpital, 75013 Paris, France.
El-Haou
Saïd
S
Balse
Elise
E
Neyroud
Nathalie
N
Vranckx
Roger
R
Coulombe
Alain
A
Hatem
Stéphane N
SN
D015761:4-Aminopyridine / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D000074288:Discs Large Homolog 1 Protein; D036681:Fluorescence Recovery After Photobleaching; D005822:Genetic Vectors; D007150:Immunohistochemistry; D051666:Kv1.5 Potassium Channel / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; C496981:KCNA5 protein, human; D051666:Kv1.5 Potassium Channel; D008565:Membrane Proteins; D026902:Potassium Channel Blockers; D011993:Recombinant Fusion Proteins; D015761:4-Aminopyridine; D011188:Potassium