Abstract
The application of high-voltage nanosecond electric pulses (nsEP) causes the formation of nanopores in plasma membrane of mammalian cells, modifies function of voltage-gated ion channels. However, it is not known if nsEPs affect ion channels directly, or these effects are mediated in alternate method. We used the whole-cell patch-clamp technique to explore the effect of 300-nsEP on voltage-gated sodium current (INa) in NG108 neuroblastoma cells. Our data have shown that a single nsEP decreased the INa in dose-dependent manners; in parallel nsEP exposures induced a non-inactivating, voltage-sensitive inward current due to nanopore formation. At the same time, the recovery of INa after nsEP exposure took significantly longer than nanopore resealing. To check if the inflow of Na+ through nanopores was efficient enough to overcome the buffering capacity of the pipette, we measured changes of Na+ concentration in “patched” cells using the Na+-sensitive fluorescent dye (Sodium Green). These experiments showed that opening of nanopores increases the Na+ concentration in patched cells; however, the maximum increase the Na+ content, even with the most intense exposure (5.3 kV/cm), was only 2.7mM, which could unlikely cause INa inhibition. The measurement of submembrane fluorescence intensity of Sodium Green by nsEP didn’t show significant increase of submembrane Na+ concentration too. The another potential pathway of reducing the INa is increasing the intracellular Ca2+ concentration after nsEP and Ca2+ mediated inhibition of INa. Our data showed that nsEP exposure in presence of high concentration Ca2+ buffer - BAPTA (20mM) in the intracellular solution caused reduce INa in NG108 cells. Our finding suggest that decreasing of INa by nsEP was not resulted of inward Na+ leakage through nanopores, as well as Ca2+ release from intracellular stores after nsEP exposure.
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