Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes.
In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.
Colinas
Olaia
O
Departamento de Bioquímica y Biología Molecular y Fisiología, Edificio IBGM, Universidad de Valladolid, C/ Sanz y Forés s/n, 47003 Valladolid, Spain.
Gallego
Mónica
M
Setién
Raúl
R
López-López
José Ramón
JR
Pérez-García
M Teresa
MT
Casis
Oscar
O
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D051672:Shal Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007093:Imidazoles; D010455:Peptides; D047428:Protein Kinase Inhibitors; D051672:Shal Potassium Channels; D013449:Sulfonamides; C514527:autocamptide-2-related inhibitory peptide II; C072105:KN 93; D000096926:Benzenesulfonamides; C031938:calmidazolium