CENTRO DE INVESTIGACIONES CARDIOVASCULARES "DR. HORACIO EUGENIO CINGOLANI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Ca2+/calmodulin dependent protein kinase II modulates the activity of the Na+-H+ exchanger in response to intracellular acidosis and phosphorylates the exchanger at noncanonical sites.
MUNDIÑA - WEILENMANN C; VILA-PETROFF M; LEZCANO N; SNABAITIS AK; VALVERDE CA; AVKIRAN M; MATTIAZZI A
Lake Las Vegas, Nevada
Conferencia; Basic Cardiovascular Sciences Conference 2009: Molecular Mechanisms of Cardiovascular Disease; 2009
basic Council of American Heart Association
Na+/H+ exchanger type 1 (NHE-1), the main pHi-controlling system activated during acidosis in the myocardium, is phosphorylated in vitro by Ca2+/calmodulin dependent protein kinase II (CaMKII). Moreover, acidosis-induced activation of CaMKII is known to contribute to the mechanical recovery from an acid load. To assess the putative role of CaMKII in the regulation of NHE-1 activity during intracellular acidosis, adult rat isolated myocytes loaded with the pHi indicator SNARF-1/AM, were subjected to an NH4Cl pulse in the absence and presence of CaMKII inhibition. Both CaMKII inhibitors, KN-93 (1mM) or the more specific AIP (1mM), slowed the rate of pHi recovery after the acid load. The modulatory role of CaMKII on NHE-1 activity was further confirmed in myocytes overexpressing CaMKII: a faster pHi recovery from intracellular acidosis was observed in CaMKII-overexpressing myocytes when compared to âgal-overexpressing myocytes (dpHi/dt: 0.195±0.04 vs. 0.045±0.010 min-1, n=8, respectively). Furthermore, myocytes isolated from transgenic mice with chronic cardiac CaMKII inhibition due to the expression of a specific inhibitory peptide (AC3-I), showed a slower rate of pHi recovery after the acid load when compared with transgenic mice expressing an inactive scrambled version of AC3-I. The stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 pathway since acidosis induced ERK1/2 phosphorylation was not abolished by the CaMKII inhibitors and simultaneous blockade of both pathways (KN-93 and PD98059) produced a further decrease in the rate of pHi recovery than when the inhibitors where given independently. Finally, in vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C terminal domain of NHE-1 indicated that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites (Ser648, Ser703 and Ser796). The results indicate a significant role of CaMKII in the stimulation of sarcolemmal NHE-1 activity in response to intracellular acidosis and suggest that this effect occurs through the phosphorylation of the exchanger at non canonical sites.