Cardiac L-type Ca Channel as an Oxygen Sensor; Possible Involvement of Ca/Calmodulin Binding Domain

Abstract

Cellular oxygen sensing is defined as the ability of a cell to respond to changes in extracellular oxygen pressure through regulation of membrane ionic currents, mitochondrial oxidative phosphorylation or transcription of regulatory proteins. In the heart hypoxia has been shown to alter ionic currents through mitchondrial redox regulation and phosphorylation by kinases. Here we report a novel oxygen sensing mechanism of cardiac L-type Ca channels which is independent of mitochondrial ROS and is partially regulated by PKA phosphorylation in the left ventricle. When oxygen pressure was locally decreased from 150 to 5 mmHg within 50 ms, an immediate suppression (25%) occurred in baseline Ica that maximized in 40-50 seconds. This response was inhibited by PKA phosphorylation on the left but not the right ventricle. Inhibiting Ca dependent inactivation using Ba2+ as the charge carrier, lead to 40% suppression of IBa within the first 5-15s of exposure. This effect was independent of PKA phosphorylation and equally affected both ventricles. Inhibiting SR Ca release with 5uM thapsigargin did not mimic the response seen with Ba2+. However, inhibiting Calmodulin using CaM inhibitory peptide 290-309 partially suppressed phosphorylated Ica in the left ventricle. This effect was also present in HEK 293 cells expressing all subunits of the recombinant L-type Ca channel. Furthermore, mutating 80 amino acids in the Ca binding/IQ domain of the alpha 1C subunit which removes Ca dependent inactivation and leads to similar kinetics of IBa and ICa, abolished the suppression of IBa under low O2. Based on these observations we propose that Cardiac L-type channels have oxygen sensing properties and that Ca/Calmodulin binding domain is a key site in this process.