A single high-intensity nanosecond electric pulse stimulates NALCN and TRPC4/5 channels leading to Na+ accumulation in adrenal chromaffin cells as detected by live cell Na+ fluorescence imaging.

Abstract

We recently reported that a single 5 ns high intensity electric pulse (NEP) elicits an inwardly rectifying current carried mainly by Na+ in bovine adrenal chromaffin cells, with ∼75% of the current being carried by both TRPC4/5 channels and the Na+ leak channel (NALCN) (Yang et al. Arch Biochem Biophys 723: 109252, 2022). In this study we used Na+ fluorescence imaging to further investigate the involvement of these channels in mediating Na+ influx triggered by NEPs. Chromaffin cells were loaded with the Na+ fluorescent indicator ING-2/AM (2 μM; Ex/Em: 525/545 nm; Kd = 20 mM). Similar to patch clamp experiments, a single NEP (5 MV/m) consistently evoked Na+ accumulation in all cells, which was characterized by a gradual rise in fluorescence that reached a plateau by ∼4 min. Also consistent with the inward current monitored by patch clamp, a combination of the TRPC4/5 channel inhibitor M084 (40 µM) and the NALCN blocker CP-96345 (50 µM) blocked the increase in fluorescence by ∼69%. Surprisingly, Na+ influx was significantly reduced in the absence of extracellular Ca2+ or by blocking L-type voltage-gated Ca2+ channels with nitrendipine, nifedipine or verapamil. The NEP-evoked Na+ influx pathway did not involve nicotinic agonist receptor stimulation nor voltage-gated Na+ channels. Overall, these results suggest that a 5-ns pulse triggers a Na+ conductance through NALCN and TRPC4/5 channels, serving as a novel way to modify neural cell excitability. Future experiments will identify the mechanism by which the NEP activates Na+ influx through these channels and whether these channels can also be activated by conventional electric stimulation (i.e., milli- and micro-second electric pulses).