LAUSR

Background: During ischemic heart disease, pH drops from 7.4 to 6.0 within 10 minutes of onset, severely affecting ion channel gating. The cardiac sodium channel (Nav1.5) is particularly susceptible to this abrupt pH change, and its altered gating is thought to predispose patients suffering ischemia to arrhythmia and sudden cardiac death. We observed the voltage-sensing domains (VSDs) of NaV1.5 to discover molecular mechanisms of its regulation by pH.Methods: A cysteine mutation was made in each of the four VSDs (DI-DIV) of Nav1.5. Synthesized RNA from these constructs was injected into Xenopus oocytes. Once channels were expressing, a fluorophore was tethered to the cysteine via a disulfide bond. By measuring the kinetics and change in magnitude of the fluorescence, we were able to track VSD conformational changes along with the current-voltage relationship.Results:Reducing the pH of the extracellular solution from 7.4 to 6.0 causes INa to decrease in magnitude by 50%, and shifts in both activation and fast inactivation rightward, consistent with previous results. At a pH of 6.0, time to peak was reduced by 300% while inactivation was only 10% slower. Observation of the VSDs showed that the DII-VSD is not affected by pH, and the DIII-VSD showed a small depolarizing activation shift ∼6.65 mV. The DIV-VSD displayed a complex phenotype, not shifting after short pulses, but shifting prominently (23.27 mV) after prolonged pulses. Its kinetics were also slowed by a factor of 2 at a pH of 6.0.Conclusions:These results suggest an important role for the DIV-VSD in determining regulation of NaV1.5 by pH.