Novel regulatory mechanisms of cardiac L-type calcium channel
Cardiac function is strongly affected by serum oxygen tension, pH and ionic gradient. These factors affect many functions of the heart including the conductance of L-type calcium channels. In this dissertation we have identified novel effects of oxygen tension and external Na+ concentration in conductance of L-type calcium channel in rat ventricular myocytes as well as the recombinant L-type channel expressed in HEK293 cells. Similar to other groups we observed that under anoxic conditions (0–5mmHg O2 tension) L-type calcium current decreased by an average of 27% within a minute. The suppressive effects of hypoxia were blocked by protein kinase A phosphorylation of the channel but was reversed and enhanced with use of H-89, inhibition of Calmodulin kinase II or use of barium as the charge carrier. Incubation with thapsigargin revealed that the anoxic sensitivity of barium transporting channel is due to loss of calcium entry through the channel and not SR calcium release. Mutational studies further revealed that loss of Calmodulin (CaM) activity or mutation of CaM binding residues of the C-tail block the ability of the channel to respond to oxygen loss. It thus appears that the CaM binding domain of the calcium channel C-tail is an immediate site of hypoxic signaling in cardiac muscle and if effectively modified can serve as a therapeutic site for prevention of calcium overload under ischemia/hypoxic conditions. In addition to oxygen deprivation we have observed that incremental reduction of extracellular sodium from 145 to 100mmol/L, suppresses calcium current by a maximum of 30%. The observed suppression was not enhanced with further reduction of external sodium to 10mmol/L nor was it dependent on channel’s phosphorylation state, but was voltage dependent and was enhanced by increasing channel mean open time, indicating an interaction of sodium ions with the channel pore. Enhanced suppressive effect of protons in presence of low external sodium further revealed that the interaction site of sodium ions is near the channel protonation site. These findings suggest that sodium ions bind/interact within the channel pore and possibly through competition with protons and other cations enhance calcium channel conductance.