Abstract
We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.
Highlights
The advent of systems to deliver nanosecond duration electric pulses (NEPs) to biological cells has sparked a great deal of interest in exploring uses of these pulses for biomedical applications, such as neurostimulation
An initial series of experiments performed in bovine chromaffin cells exposed to normal K+based external (BSS) and internal solutions was carried out to determine the ionic nature of the early inward current elicited by depolarizing voltage clamp steps from a holding of –70 mV
As reported in this study, we found that application of 5 ns pulses as a pulse pair can modulate voltage-gated Na+ channels in a unique manner, which points to the potential for NEPs to influence overall cell excitability in novel ways
Summary
The advent of systems to deliver nanosecond duration electric pulses (NEPs) to biological cells has sparked a great deal of interest in exploring uses of these pulses for biomedical applications, such as neurostimulation. An example of the potential use of NEPs for neurostimulation is the report by Jiang and Cooper [1] that a single 12 ns pulse was capable of activating skin nociceptors. Previous studies from our group have shown that a 5 ns pulse can stimulate catecholamine release in neuroendocrine adrenal chromaffin cells by causing Ca2+ influx via voltage-gated calcium channels (VGCCs) [3]. 12 ns pulse exposure triggers Na+ influx via voltagegated Na+ channels, which is responsible for the generation of action potentials [2]. In contrast, Na+ influx via voltage-gated Na+ channels is not responsible for the membrane depolarization that evokes VGCC activation in cells exposed to a 5 ns pulse [4]. A 5 ns pulse causes an inhibition of voltage-gated Na+ channels in these cells [6]
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