色素性乾皮病患者ノ「デモンストラチヲン」及ビ色素性乾皮病ノ一斑

<p>(A) Comparison of peak current density (pA/pF) of inward currents elicited by 40 ms voltage steps from -140 mV to -40 mV in HEK-293 cells stably expressing Nav1.9 alone or coexpressed with β1 and β2 subunits in presence and absence of 500 μM GTP-γ-S in intracellular solution. (B) Representative family current traces recorded in HEK-293 cells expressing Nav1.9 + β1/β2 recorded with 500 μM GTP-γ-S in pipette following a series of incremental voltage steps from –140 mV to +40 mV. (C) Inward current traces elicited in Nav1.9 + β1/β2 by 40 ms voltage steps from -140 mV to -40 mV in extracellular buffer containing 135 mM Na⁺ in absence or presence 10 μM tetrodotoxin (TTX) or in an extracellular buffer where all the sodium was replaced by choline. (D) Current density voltage relationship recorded in HEK-293 cells stably expressing Nav1.9 + β1/β2 + 500 μM GTP-γ-S (data are mean ± SEM from n = 13 observations). (E) Voltage dependence of activation and inactivation of Nav1.9 + β1/β2 recorded in the presence and absence of 500 μM GTP-γ-S following incremental depolarizing 100 ms conditioning voltage steps from -140 mV followed by a 40 ms test pulse to -40 mV. Data was fitted to a Boltzmann equation with parameters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t002" target="_blank">Table 2</a>. (F) Time course of recovery inactivation induced by either a 100 ms (circle) or 5 s (triangle) depolarizing voltage step to 0 mV. Recovery was assessed by applying a 40 ms test pulse to -40 mV after variable periods at -140 mV. Data was fit with a two phase exponential equation with fitted fast and slow time constants shown in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t004" target="_blank">4</a>.</p

Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells

<div><p>The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.</p></div

Potencies for Inhibition of Nav1.7 and Nav1.8.

<p>Potencies for Inhibition of Nav1.7 and Nav1.8.</p

Pharmacological modulation of human Nav1.9.

<p>(A) Magnitude of Nav1.9 inactivation at different holding potentials. Percent inactivation determined from decline in test pulse current amplitude applied every 20s from test holding potential to -40 mV (values are mean ± SEM for 11–12 observations). (B) Comparison of concentration dependence of Nav1.9 inhibition by tetracaine at different holding potentials. (C) Representative current traces recorded in HEK-hNav1.9 cell line in the presence and absence of 3 μM TC-N 1752 (structure shown) during a 40 ms voltage step from -120 mV to -40 mV. (D) Diary plot of Nav1.9 current amplitude recorded using 40 ms voltage steps to -40 mV applied at 0.05 Hz, initially from a holding potential of -140 mV, then at -120 mV to induce ~25% inactivation. After current amplitude was stable at -120 mV, test agent was applied. Washout of test agent at holding potential of -140 mV until current amplitude became stable and then holding potential reset to -120 mV to allow inhibition to be determined by dividing current amplitude in presence of compound by interpolated current amplitude (see dotted line) between prior to compound addition and after washout. (E) Concentration response relationships for inhibition of Nav1.9 currents by known Nav channel inhibitors TC-N 1752, tetracaine, mexiletine and benzocaine. IC₅₀s for these agents are plotted in (F) (values are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t005" target="_blank">Table 5</a>) and are compared to IC₅₀s for inhibition of Nav1.7 and Nav1.8 (values are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t006" target="_blank">Table 6</a>).</p

Potencies for Inhibition of Rat and Mouse Nav1.9.

<p>Potencies for Inhibition of Rat and Mouse Nav1.9.</p

Time Constants for Recovery from Inactivation Induced by 100 ms Prepulse to 0 mV.

<p>Time Constants for Recovery from Inactivation Induced by 100 ms Prepulse to 0 mV.</p

Characterization of K799 residue unique to Nav1.9 in Domain 2 S6.

<p>(A) Upper panel; sequence alignment of D2 S6 for Nav1.1 to Nav1.9. Lysine residue K799 is labeled in blue whereas the consensus asparagine found at the equivalent position in other Nav channel subtypes is shown in green. Lower panel; sequence alignment of Nav1.9 D2 S6 with D4 S6 segments showing position of K799 residue relative to F1592 and Y1599 residues that form part of the canonical local anesthetic binding site. (B) Homology model of human Nav1.9 based on X-ray crystal structure of NavM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.ref026" target="_blank">26</a>] illustrating the position of lysine K799 in relation to F1592 and Y1599. (C) Comparison of the voltage dependence of activation (Nav1.9 WT: -51 ± 1 mV, n = 9; K799N: -48 ± 1 mV, n = 14; P>0.05) and inactivation induced by 500ms conditioning prepulse (Nav1.9 WT: -50 ± 1 mV, n = 9; K799N: -54 ± 1 mV, n = 14; P>0.05). (D) Comparison of concentration dependence of inhibition of Nav1.9 WT and Nav1.9 K799N channels by tetracaine, TC-N 1752 and benzocaine. Potency was determined using protocol described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.g002" target="_blank">Fig 2</a>. (E) Comparison of the voltage dependence of activation and inactivation of Nav1.7 WT and Nav1.7 N945K mutant channels (activation; WT: -26 ± 0.4 mV, n = 10; N945K: -26 ± 0.4 mV, n = 9; P>0.05; and inactivation, WT: -65 ± 0.3 mV, n = 10; N945K: -64 ± 0.3 mV, n = 9; P>0.05) using same conditioning prepulse protocol as in (C) except utilizing a 20 ms test pulse to 0 mV. (F) Comparison of concentration dependence of inhibition of Nav1.7 WT and Nav1.7 N945K channels by tetracaine, TC-N 1752 and benzocaine.</p

Potencies for Inhibition of Nav1.9 and Nav1.9 F1592A/Y1599A.

<p>Potencies for Inhibition of Nav1.9 and Nav1.9 F1592A/Y1599A.</p

Interaction of sodium channel inhibitors with Nav1.9 canonical local anesthetic binding site.

<p>(A) Sequence alignment of D4 S6 segments for Nav1.1 to Nav1.9. The canonical local anesthetic binding site residues phenylalanine and tyrosine are labeled in magenta and were mutated to alanine (F1592A/Y1599A) to assess their contribution to Nav1.9 inhibition. (B) Comparison of the voltage dependence of activation (Nav1.9 WT: -51 ± 1 mV, n = 9; Nav1.9 F1592A/Y1599A: -56 ± 1 mV, n = 10; P>0.05) and inactivation following a 500 ms conditioning prepulse (WT: -50 ± 1 mV, n = 9; Nav1.9 F1592A/Y1599A: -56 ± 1 mV, n = 10; P<0.05). Comparison of concentration response relationships for inhibition of Nav1.9 WT (open symbols) and Nav1.9 F1592A/Y1599A (Closed symbols, LAM) by tetracaine (C), TC-N 1752 (D), and benzocaine (E). Fitted IC₅₀s are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161450#pone.0161450.t005" target="_blank">Table 5</a>.</p

Current Density of HEK-293 hNav1.9 Stable Cell Lines.

<p>Current Density of HEK-293 hNav1.9 Stable Cell Lines.</p