Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo

Luis F Lopez-Santiago,William J Brackenbury,Chunling Chen,Lori L Isom

doi:10.1074/jbc.m111.242370

Luis F Lopez-Santiago, William J Brackenbury + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.242370

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Abstract

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).

Highlights

All four mammalian VGSC ␤ subunit gene products are expressed in dorsal root ganglion (DRG) neurons [4]; little is known about
Using a well established voltage clamp protocol based on previously observed differences in voltage gating properties between TTX-S and TTX-R VGSCs expressed in DRG neurons [4, 20], two types of INa were dissected from the total INa
The overall conclusion of this work is that Scn1b null nociceptive neurons are hyperexcitable, suggesting that Scn1b plays an important role in maintaining normal excitability and, with that pain sensation, in WT DRGs

Summary

EXPERIMENTAL PROCEDURES

Preparation of DRG Neurons—The generation of Scn1b null mice was described previously [13]. Each data set (a plot of peak INa during the 0 mV test pulse versus prepulse voltage) was fit with the summation of two Boltzmann equations of the following form, INa ϭ F1/͑1 ϩ exp͑Vm Ϫ V11/ 2͒/k1͒͒ ϩ F2/͑1 ϩ exp͑Vm Ϫ V21/ 2͒/k2͒͒ (Eq 2). Isolated Kϩ Currents (IK)—IK were recorded from single, small DRG neurons at RT in the presence of a bath solution that contained 150 mM choline-Cl, 5 mM KCl, 1 mM MgCl2, 0.1 mM CdCl2, 10 mM HEPES, 10 mM glucose (pH 7.3 with KOH), and 1 ␮M TTX. Ten images each of DRG sections colabeled with anti-Nav1.8 or anti-Nav1.9 and anti-peripherin were analyzed per mouse, for a total of three WT and three null mice. Signal intensity in the plasma membrane region, defined as 0.75 ␮m inward from the outer edge of labeling, was compared with average cytoplasmic signal intensity of equivalent internal length

RESULTS

Slow inactivation parameters from the double exponential fit of INa availability

DISCUSSION