Nitric Oxide Potentiates the Effect of Calcium/Calmodulin-Dependent Kinase II on Ca v 1.2 Protein

Abstract

The L-type calcium channel (LTCC) is the major route for calcium influx into cardiac myocytes. Ca2+/calmodulin-dependent kinase II (CaMKII) facilitates calcium influx by binding to the C terminal domain of the Cav1.2 protein (main pore forming ion conducting subunit of the LTCC). Nitric oxide (NO) is proposed to bind to the LTCC and alter calcium influx. However, it is not known whether this occurs as a result of a direct effect of NO on the channel. The aim of this study was to clarify the effect of NO and CaMKII on Cav1.2 protein function. We expressed the human long N terminal (NT) isoform of Cav1.2 in HEK cells. The channel protein was purified by histidine tag purification and incorporated in liposomes for functional analysis by patch-clamp technique. Exposing the long NT isoform to the NO donor GSNO or sodium nitroprusside failed to increase channel open probability (relative Po 0.696 ± 0.153, n = 5 and 1.09 ± 0.02, n = 6 respectively). Application of CaMKII alone increased open probability (Po) of the long NT isoform 2.24 ± 0.16 fold (n = 6; p < 0.05). The effect was more pronounced when CaMKII was pre-exposed to the NO donor (2.98 ± 0.13 fold, n = 6; p < 0.05), and these conditions resulted in a significant increase in CaMKII activity as measured by our FRET reporter (relative activity 1.36 ± 0.08 vs. baseline, n = 6; p < 0.05). The results suggest that CaMKII, but not the channel itself, is sensitive to nitrosylation. We conclude that the function of Cav1.2 protein cannot be directly altered by NO, but the functionally relevant target of NO is CaMKII.