Bosmans F et al. (2011), Functional properties and toxin pharmacology of...

XB-ART-44237

J Gen Physiol 2011 Jul 01;1381:59-72. doi: 10.1085/jgp.201110614.

Show Gene links Show Anatomy links

Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors.

Bosmans F , Puopolo M , Martin-Eauclaire MF , Bean BP , Swartz KJ .

???displayArticle.abstract???
The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.

???displayArticle.pubmedLink??? 21670206
???displayArticle.pmcLink??? PMC3135324
???displayArticle.link??? J Gen Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: drg1 gnl3 kcnb1 nav1 tbx2

???attribute.lit??? ???displayArticles.show???

	Figure 1. Transfer of the voltage sensor paddle motifs from hNav1.9 to Kv2.1. (A) Cartoon representing a top view of Nav1.9 (left) and Kv2.1 (right). The central Na+- or K+-selective pore is surrounded by the four voltage sensors of the four domains (DI–DIV, delineated by the dotted line). In Nav1.9, the paddle motifs are not identical and are therefore colored differently: purple, domain I paddle (DI); red, domain II paddle (DII); blue, domain III paddle (DIII); green, domain IV paddle (DIV). This color code is used in the other figures. In Kv2.1, the paddle motifs are identical and therefore have the same color (gray). (B and C) Transfer of the hNav1.9 paddle motifs into Kv2.1. Families of potassium currents (B) and tail current voltage–activation relationships (C) for each chimeric construct (n = 16; error bars represent SEM). Holding voltage was −90 mV, and the tail voltage was −50 mV (−80 mV for DII). Bars in B are 1 µA and 100 ms.
	Figure 2. Kinetics of opening and closing for hNav1.9/Kv2.1 chimeric constructs. (A) Representative macroscopic currents showing channel activation (left) and channel deactivation (right) using the following voltage protocols: activation, 10-mV incrementing steps to voltages between −60 and +100 mV (−90 mV for DII) from a holding potential of −90 mV; deactivation, 10-mV incrementing steps to voltages between −10 and −100 mV (−150 mV for DII) from a test voltage of between +80 and +100 mV (holding potential is −90 mV). (B) Mean time constants (τ) from single-exponential fits to channel activation (filled circles) and deactivation (open circles) plotted as a function of the voltage at which the current was recorded. n = 4–8, and error bars represent SEM.
	Figure 3. Sensitivity of hNav1.9/Kv2.1 constructs to extracellular toxins. Effects of toxins on Kv2.1 and chimeric constructs where paddle motifs were transferred from hNav1.9 into Kv2.1. Normalized tail current voltage–activation relationships are shown where tail current amplitude is plotted against test voltage before (black) and in the presence of toxin (other colors). Data are grouped per toxin (horizontally) and per chimera or wild-type Kv2.1 (vertically). Concentrations used are 100 nM PaurTx3, ProTx-I, ProTx-II, and TsVII, and 1 µM AaHII. The holding voltage was −90 mV, the test pulse duration was 300 ms, and the tail voltage was −50 mV (−80 mV for DII). n = 3–5, and error bars represent SEM.
	Figure 4. Sensitivity of rNav1.9/Kv2.1 constructs to extracellular toxins. (A) Families of potassium currents, (B, left) tail current voltage–activation relationships, and (B, right) kinetics of opening and closing for Kv2.1 channels containing paddle motifs from the four domains of rNav1.9 for each chimeric construct (n = 8, and error bars represent SEM). Holding voltage was −90 mV, and the tail voltage was −60 mV. Bars in A are 0.5 µA and 100 ms (200 ms for DII and DIV). Mean time constants (τ) from single-exponential fits to channel activation (filled circles) and deactivation (open circles) are plotted as a function of the voltage at which the current was recorded. (C) Effects of TsVII and ProTx-I on Kv2.1 and chimeric constructs where paddle motifs were transferred from rNav1.9 into Kv2.1. Normalized tail current voltage–activation relationships are shown where tail current amplitude is plotted against test voltage before (black) and in the presence of toxin (other colors). Concentration used is 100 nM. Apparent Kd of TsVII for the DII construct is 202 ± 6 nM. Apparent Kd of ProTx-I for DI, DIII, and DIV is 412 ± 8 nM, 77 ± 21 nM, and 133 ± 36 nM, respectively (calculated Kd assuming four independent toxin-binding sites per channel). The holding voltage was −90 mV, the test pulse duration was 300 ms, and the tail voltage was −60 mV. n = 3–5, and error bars represent SEM.
	Figure 5. Effect of TsVII on rNav1.8. (A) rNav1.8 currents at +15 mV are not affected by 100 nM TsVII (holding voltage was −90 mV). (B) G-V relationship of rNav1.8 before (black) and after (green) the application of 100 nM TsVII. n = 3, and error bars represent SEM. (C) rNav1.8 currents at +15 mV are only slightly inhibited when 1 µM TsVII is applied in combination with a preceding 5-ms depolarizing pulse to +30 mV (holding voltage was −90 mV) (Cestèle et al., 1998). (D) Deduced G-V relationship of rNav1.8 before (black) and after (green) the application of 100 nM TsVII. Open circles indicate the use of a depolarizing prepulse. n = 3, and error bars represent SEM.
	Figure 6. Inhibition of rNav1.8 by ProTx-I. (A) Inhibition of rNav1.8 currents by 100 nM ProTx-I at +15 mV (holding voltage was −90 mV). (B and C) Current–voltage relationship (B) and deduced G-V relationship (C) of rNav1.8 before (black) and after (green) the application of 100 nM ProTx-I. n = 3–5, and error bars represent SEM. (D) Concentration dependence for ProTx-I inhibition of rNav1.8 plotted as fraction unbound (Fu) measured at negative voltages. Dotted line is a fit of the Hill equation to the data (IC50 = 10.9 ± 0.5 nM; slope = 1.3 ± 0.1). Solid line is a fit with a three-binding site model (Kd = 45.1 ± 2.7 nM). See Materials and methods for more information. (E) Effect of 100 nM ProTx-I on Kv2.1 channels containing paddle motifs from rNav1.8. Normalized tail current voltage–activation relationships are shown before and after the application of the toxin. n = 4, and error bars represent SEM.
	Figure 7. Effect of TsVII on native Nav1.9 currents in rat DRG neurons. (A) Enhancement by 100 nM TsVII of TTX-r current evoked by steps to voltages between −55 and −35 mV. Control currents were recorded with a steady holding potential of either −80 mV (black traces) or −120 mV (blue traces), established for >5 min to remove inactivation from rNav1.9 channels. In both cases, the test pulse was preceded by a 400-ms step to −140 mV. 100 nM TsVII (green traces) was applied at the holding potential of −120 mV. (B) Peak conductance versus test voltage before and after 100 nM TsVII (different cell than A). Closed symbols, peak conductance calculated from peak current using a reversal potential of +50 mV; smooth curves, best fits to a Boltzmann function according to G = Gmax/(1 + e−(V − V1/2)/k), where Gmax is the maximal conductance, V1/2 is the half-activation voltage, and k is the slope factor. Control: Gmax = 25 nS, V1/2 = −34 mV, and k = 4.9 mV. 100 nM TsVII: Gmax = 174 nS, V1/2 = −54 mV, and k = 2.3 mV. (C) Voltage dependence of inactivation determined using a test pulse to −50 mV from a variety of holding potentials established for 2 s. Smooth curves are best fits to a Boltzmann function according to: I/Imax = (1 + e(V − V1/2)/k)−1, where I/Imax is the normalized peak conductance, V1/2 is the midpoint of inactivation, and k is the slope factor. Control: V1/2 = −45 mV and k = 6.2 mV. 100 nM TsVII: V1/2 = −55 mV and k = 3.6 mV. All solutions contained 300 nM TTX to block TTX-s Nav channels.
	Figure 8. Effect of ProTx-I on native Nav1.9 currents in rat DRG neurons. (A) Enhancement by 100 nM ProTx-I of rNav1.9-mediated current evoked by same protocol as in Fig. 7 A. (B) Peak conductance versus test voltage before and after 100 nM ProTx-I (different cell than A). Closed symbols, peak conductance calculated from peak current using a reversal potential of +50 mV; smooth curves, best fits to a Boltzmann function, as in Fig. 7 B. Control: Gmax = 70 nS, V1/2 = −35 mV, and k = 5.0 mV. 100nm ProTx-I: Gmax = 49 nS, V1/2 = −44 mV, and k = 6.0 mV. (C) Voltage dependence of inactivation determined using a test pulse to −50 mV from a variety of holding potentials established for 2 s as in Fig. 7 C. Smooth curves are best fits to Boltzmann functions. Control: V1/2 = −41 mV and k = 6.2 mV. 100 nM ProTx-I: V1/2 = −47 mV and k = 5.6 mV. All solutions contained 300 nM TTX to block TTX-s Nav channels.
	Figure 9. Effect of TsVII and ProTx-I on native Nav1.8 currents in rat DRG neurons. (A) Effect of 100 nM TsVII on rNav1.8-mediated current, evoked by a step from a holding potential of −50 mV in the presence of 300 nM TTX. (B) Effect of 100 nM ProTx-I on native rNav1.8-mediated current.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase