1 2 Regulation of Nav 1 .

40 Voltage-gated sodium (Na) channels are composed of a pore-forming α subunit and one 41 or more auxiliary β subunits. The present study investigated the regulation by the β 42 subunit of two Na channels (Nav1.6, Nav1.8) expressed in dorsal root ganglion (DRG) 43 neurons. Single-cell RT-PCR was used to show that Nav1.8, Nav1.6, and the β1-β3 44 subunits were widely expressed in individually harvested small-diameter DRG neurons. 45 Co-expression studies were used to assess the regulation of Nav1.6 and Nav1.8 by β 46 subunits. The β1 subunit induced a 2.3-fold increase in Na current density and 47 hyperpolarizing shifts in the activation (–4 mV) and steady-state inactivation (–4.7 mV) 48 of heterologously expressed Nav1.8 channels. The β4 subunit caused more pronounced 49 shifts in activation (–16.7 mV) and inactivation (–9.3 mV) but did not alter the current 50 density of cells expressing Nav1.8 channels. The β3 subunit did not alter Nav1.8 gating 51 but significantly reduced the current density by 31%. This contrasted with Nav1.6, where 52 the β subunits were relatively weak regulators of channel function. One notable exception 53 was the β4 subunit, which induced a hyperpolarizing shift in activation (–7.6 mV) but no 54 change in the inactivation or current density of Nav1.6. The β subunits differentially 55 regulated the expression and gating of Nav1.8 and Nav1.6. To further investigate the 56 underlying regulatory mechanism, β subunit chimeras containing portions of the strongly 57 regulating β1 and weakly regulating β2 subunit were generated. Chimeras retaining the C58 terminal domain of β1 produced hyperpolarizing shifts in gating and increased the current 59 density of Nav1.8 similar to that observed for wild-type β1 subunits. The intracellular C60 terminal domain of β1 appeared to play an essential role in the regulation of Nav1.8 61 expression and gating. 62 63