Abstract
Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9) was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNav1.9. By using the hNav1.9 expression system, we investigated the electrophysiological properties of four mutations of hNav1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNav1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNav1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNav1.9 activity, indicating its vital role in hNav1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNav1.9 studies and new insight into mechanism of hNav1.9 linking to pain.
Highlights
Voltage-gated sodium channels (VGSCs) are a cluster of important transmembrane proteins, that play crucial roles for initiation and propagation of action potential (AP) in excitable tissues, including heart, brain and peripheral nerves (Catterall, 2000, 2012)
It exhibits unique biophysical properties that include activation near resting membrane potential, slow activation and inactivation kinetics, generation of large persistent currents in low depolarizing voltages and large window current (Cummins et al, 1999; Dib-Hajj et al, 2002, 2015). These make Nav1.9 act as a threshold channel in AP firing, amplifying sub-threshold stimulus that leads to AP burst and increases repetitive firing (Herzog et al, 2001; Dib-Hajj et al, 2002, 2015)
We succeeded in achieving a functional expression of hNav1.9 channel in heterologous cells by fusing GFP to the C-terminal of hNav1.9 sequence, which provides a powerful platform for the electrophysiological studies of hNav1.9. By applying this expression system, we systematically investigated the biophysical characteristics of wild type (WT) and mutant hNav1.9 channels, as well as elucidated the effects and mechanisms of inflammatory mediators on hNav1.9
Summary
Voltage-gated sodium channels (VGSCs) are a cluster of important transmembrane proteins, that play crucial roles for initiation and propagation of action potential (AP) in excitable tissues, including heart, brain and peripheral nerves (Catterall, 2000, 2012). It exhibits unique biophysical properties that include activation near resting membrane potential, slow activation and inactivation kinetics, generation of large persistent currents in low depolarizing voltages and large window current (Cummins et al, 1999; Dib-Hajj et al, 2002, 2015). These make Nav1.9 act as a threshold channel in AP firing, amplifying sub-threshold stimulus that leads to AP burst and increases repetitive firing (Herzog et al, 2001; Dib-Hajj et al, 2002, 2015)
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