Co-expression of Na_Vβ subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins

Abstract

Background and Purpose

Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (Na_V1) and one or two accessory β-subunits (Na_Vβs). Neurons in mammals can express one or more of seven isoforms of Na_V1 and one or more of four isoforms of Na_Vβ. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in Na_V1. Hitherto, the effects of Na_Vβ-subunit co-expression on the activity of these toxins have not been comprehensively assessed.

Experimental Approach

Four μ-conotoxins (μ-TIIIA, μ-PIIIA, μ-SmIIIA and μ-KIIIA), TTX and STX were tested against Na_V1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of Na_Vβ1, β2, β3 or β4 and, for Na_V1.7, binary combinations of thereof.

Key Results

Co-expression of Na_Vβ-subunits modifies the block by μ-conotoxins: in general, Na_Vβ1 or β3 co-expression tended to increase k_on (in the most extreme instance by ninefold), whereas Na_Vβ2 or β4 co-expression decreased k_on (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by Na_Vβ-subunit co-expression. Tests of Na_Vβ1 : β2 chimeras co-expressed with Na_V1.7 suggest that the extracellular portion of the Na_Vβ subunit is largely responsible for altering μ-conotoxin kinetics.

Conclusions and Implications

These results are the first indication that Na_Vβ subunit co-expression can markedly influence μ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. μ-Conotoxins could, in principle, be used to pharmacologically probe the Na_Vβ subunit composition of endogenously expressed VGSCs.

Introduction

Voltage-gated sodium channels (VGSCs), which are responsible for the upstroke of the action potential, consist of two classes of integral membrane glycoprotein subunits, large (260 kDa) α-subunits and smaller (30–40 kDa) β-subunits. These subunits assemble into complexes formed from a single α-subunit and one or two β-subunits. Mammals have nine isoforms of the α-subunit (Na_V1.1 through 1.9) and four of the β-subunit (Na_Vβ1 through β4) (Catterall et al., 2005). The α-subunit, which comprises four homologous domains, each with six membrane-spanning segments, bears the essential features of a functioning VGSC; namely, a Na⁺-selective pore as well as voltage-sensors responsible for the gating of the channel in response to changes in membrane potential [for recent review see (Catterall, 2012)]. The β-subunit has a single membrane-spanning segment, with a large extracellular and small intracellular domain, and regulates the expression and trafficking of the α-subunit as well as modulates its voltage sensitivity (for recent reviews see Brackenbury and Isom, 2011; Chahine and O'leary, 2011). Na_Vβ2 and β4 are covalently linked to the α-subunit via a disulfide bond (Isom et al., 1995a; Yu et al., 2003), whereas Na_Vβ1 and β3 are non-covalently linked (Isom et al., 1992; Morgan et al., 2000). Multiple α-subunit paralogs have been found in all vertebrates examined (Lopreato et al., 2001; Widmark et al., 2011); and conserved orthologs of all four mammalian Na_Vβ isoforms are found in fish, frog and bird, with Na_Vβ1/β3 and Na_Vβ2/β4 sharing a common ancestry (Chopra et al., 2007).

Adult rat dorsal root ganglia (DRG) consist of a heterogeneous population of sensory neurons, where more than one Na_V1 and Na_Vβ isoform can be expressed by a given neuron (see reviews by Dib-Hajj et al., 2010; Chahine and O'leary, 2011). Thus, a given neuron may express a multiplicity of different species of VGSCs, each composed of different combinations of Na_V1 and Na_Vβ isoforms, and to untangle this mélange by pharmacological means presents a challenge. We are attempting to address this problem by using conotoxins that target VGSCs, of which there are four families: μ-, μO-, δ- and ι-conotoxins. Each family has a characteristic amino acid sequence framework and distinct mode of action: μ-conotoxins are pore blockers, whereas the other three families consist of gating modifiers (Terlau and Olivera, 2004; Fiedler et al., 2008; Lewis et al., 2012).

We recently reported that the action potentials in A and C fibres in rat sciatic nerve could be pharmacologically dissected with a panel of μ-conotoxins, whose Na_V1 isoform selectivities were determined for Na_V1.1 through 1.8 expressed in Xenopus laevis oocytes. We concluded that mainly Na_V1.6 and Na_V1.7 were the responsible for the propagation of action potentials in A and C fibres respectively (Wilson et al., 2011a). In the course of investigating the block by μ-conotoxins of TTX-sensitive sodium currents (TTX-s I_Na) of voltage-clamped, acutely dissociated rat dorsal root ganglion (DRG) neurons, we observed an inconsistency insofar as the kinetics of block by μ-SmIIIA of what we surmised were sodium currents mediated by Na_V1.7 differed between large neurons and a subset of small neurons (Zhang et al., 2013). Thus, we were motivated to investigate whether association of Na_V1.7 with different Na_Vβ subunits might account this discrepancy. In this report, we examined four TTX-sensitive Na_V1 isoforms reported to be normally present in adult rat DRG neurons, Na_V1.1, 1.2, 1.6 and 1.7 (Black 1996; 2004; Rush et al., 2007; Fukuoka et al., 2008), and compared how their co-expression with each of the four rat Na_Vβ subunits in X. laevis oocytes affected their block by three conotoxins, μ-TIIIA, μ-PIIIA and μ-SmIIIA, whose sequences are illustrated in Figure 1. These conotoxins had been previously tested against Na_V1.1-1.8 (all from rat except Na_V1.6, which was from mouse) expressed in oocytes without co-expression of any Na_Vβ subunit (Wilson et al., 2011a). In this report, all Na_V1 subunits examined were from rat, including Na_V1.6, whose sensitivity to μ-conotoxins, are reported here for the first time.

Figure 1.

Amino acid sequences of the four μ-conopeptides used in this report. Sequences were aligned by their Cys residues, which are in bold. Z = pyroglutamine, O = hydroxyproline, # = amidated carboxyl terminal. References for sequences are as follows: μ-KIIIA (Bulaj et al., 2005); μ-PIIIA (Shon et al., 1998); μ-SmIIIA (West et al., 2002) and μ-TIIIA (Lewis et al., 2007).

Particular attention was devoted to Na_V1.7 because this Na_V1 isoform is implicated in the pathophysiology of inherited and acquired pain states, and antagonists of Na_V1.7 could serve as analgesics (Dib-Hajj et al., 2009a; 2010). Only 2 of 11 μ-conotoxins recently examined blocked Na_V1.7 with IC₅₀ or K_d ≲ 1 μM: μ-SmIIIA and μ-KIIIA (Wilson et al., 2011a); thus, in addition to μ-SmIIIA, μ-KIIIA was also tested on Na_V1.7 co-expressed with the various Na_Vβ subunits. The sequence of μ-KIIIA is also illustrated in Figure 1.

We show here that co-expression of Na_Vβ subunits does alter the affinities of μ-conotoxins. We believe this is the first demonstration that co-expression of Na_Vβ subunits can affect the binding of a toxin that blocks VGSCs by interacting with the extracellular vestibule of the pore of the channel; that is, neurotoxin receptor site 1 (Cestèle and Catterall, 2000). Tetrodotoxin (TTX) and saxitoxin (STX), which are guanidinium alkaloids, were originally used to define site 1 (Catterall, 1980); thus, we also examined these two alkaloids and report here that their activities were minimally, if at all, altered by Na_Vβ subunit co-expression. This is an interesting result to contemplate in view of our recent findings that the μ-conotoxin binding site appears to abut that of TTX and STX, but is situated more superficially in the vestibule (Zhang et al., 2009; 2010a).

Methods

Toxins

μ-Conotoxins were synthesized as previously described (Wilson et al., 2011a). TTX was purchased from Alomone Labs (Jerusalem, Israel) and STX from the National Research Council of Canada (Halifax, Nova Scotia, Canada).

Cloning of rat Na_V1.6

Rat Na_V1.6 DNA (GenBank accession #NM_019266.2), subcloned in pSGEM vector (which was derived from pGEMHE vector) (Liman et al., 1992), was synthesized by GenScript USA (Piscataway, NJ, USA). The DNA was amplified using the GenomiPhi V2 DNA amplification kit (GE Lifesciences, Pittsburgh, PA, USA). The amplified DNA was sequenced, digested with NheI and transcribed with T7 RNA polymerase (mMessage mMachine RNA transcription kit, Ambion, Life Technology, Grand Island, NY, USA).

The remaining clones, also from rat, were obtained as follows. Na_V1.1, Na_V1.2, Na_Vβ1 and Na_Vβ2 were provided by Prof Alan A Goldin; Na_Vβ3 and Na_Vβ4 by Prof Lori L Isom; and Na_V1.7 by Prof Gail Mandel. The preparation of RNA from these was as previously described (Wilson et al., 2011a). The nomenclature of the channel subunits conforms to this journal's Guide to Receptors and Channels (Alexander et al., 2011).

Construction of Na_Vβ chimeras Na_Vβ112 and Na_Vβ211

We followed the lead of Zimmer and Benndorf (2002) in the construction and nomenclature of these chimeras; see also (McCormick et al., 1999). Both chimeras were made by PCR. To synthesize the Na_Vβ112 chimera (which consisted of the extracellular and transmembrane portions of Na_Vβ1 and intracellular portion of Na_Vβ2) and the Na_Vβ211 chimera (which consisted of the extracellular region of β2 and the transmembrane and intracellular portions of β1), primers were designed to PCR-amplify the desired area of one subunit, followed by a 15–20 bp overhang belonging to the other subunit (designating the chimera junction; see cartoon in Figure 4B). In a subsequent PCR, the two DNA pieces were allowed to hybridize first at the overhangs and then were amplified using primers at the 5′ and 3′ ends, used to introduce restriction sites NotI and XhoI respectively. The PCR product was gel-extracted and purified using Qiaquick PCR purification kit (Qiagen Sciences, Valencia, CA, USA). The chimeras were subcloned into the pSGEM oocyte expression vector (which contains the 5′ and 3′ Xenopus globin regions) using the NotI and XhoI restriction sites, transformed into DH10B competent cells and grown in ampicillin-containing LB; DNA was isolated using Qiaprep Spin mini prep kit (Qiagen Sciences). The DNA was linearized using NheI, and sense RNA was transcribed using T7 polymerase (mMessage mMachine RNA transcription kit, Ambion).

Figure 4.

Peptide concentration dependency of the observed rate constants (k_obs) for the block by μ-SmIIIA of Na_V1.7 either alone, co-expressed with Na_Vβ1, β2, β3 or β4 (individually or in pairs) or co-expressed with the chimeras Na_Vβ112 or Na_Vβ211. Peak I_Na of voltage-clamped oocytes expressing Na_V1.7 ± co-expression of indicated Na_Vβ subunit(s) were monitored as in Figure 3. (A) The three steepest curves, which essentially overlie each other, are those of Na_Vβ1, β3 and the β112 chimera. The remaining curves, in the order of decreasing slope, are those of Na_V1.7 alone, +β3 + β2, +β1 + β2, +β3 + β4, +β211 chimera, +β1 + β4, +β2 and +β4. Slopes of curves such as these provided k_on values in Table 2. (B) Cartoon of chimeras of Na_Vβ1 and Na_Vβ2 with amino acid residues at splice sites, as well as at N- and C-termini, indicated. Top, the Na_Vβ112 chimera consisted of the extracellular and transmembrane portions of Na_Vβ1 and the intracellular portion of Na_Vβ2. Bottom, the Na_Vβ211 chimera was formed from the extracellular portion of Na_Vβ2 and the transmembrane and intracellular portions of Na_Vβ1. cRNA encoding these chimeras were made as described in Methods.

Preparation and recording from X. laevis oocytes

Use of X. laevis frogs, which provided oocytes for this study, followed protocols approved by the University of Utah Institutional Animal Care and Use Committee that conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).

Oocytes were prepared and voltage-clamped essentially as previously described (Zhang et al., 2010a; Wilson et al., 2011a). Briefly, a given oocyte was injected with 30–50 nl of cRNA in distilled water of one of the following rat Na_V1 isoforms without or with an equal weight of rat Na_Vβ isoform cRNA (when two different Na_Vβ isoforms were involved, the weight of each was equal to that of the Na_V1 isoform): Na_V1.1, Na_V1.2, Na_V1.6 or Na_V1.7 (3, 1.5, 30 or 15 ng, respectively) and incubated 1 to 6 days at 16°C in ND96 composed (in mM) of: NaCl (96), KCl (2), CaCl₂ (1.8) MgCl₂ (1) and HEPES (5), pH 7.5. The incubation medium also contained the antibiotics penicillin (100 units·mL⁻¹), streptomycin (0.1 mg·mL⁻¹), amikacin (0.1 mg·mL⁻¹) and Septra (0.2 mg·mL⁻¹). Oocytes in ND96 were two-electrode voltage-clamped using microelectrodes containing 3 M KCl (<0.5 MΩ) and clamped at a holding membrane potential of −80 mV unless indicated otherwise. Sodium channels were activated by stepping the potential to −10 mV for 50 ms every 20 s in all experiments, including toxin wash-in and washout. Current signals were filtered at 2 KHz, digitized at a sampling frequency of 10 KHz and leak-subtracted by a P/8 protocol using in-house software written in LabVIEW (National Instruments, Austin, TX, USA). All experiments were done at room temperature.

Application and washout of μ-conotoxins, TTX and STX

The oocyte-recording chamber was a 30 μL cylindrical well, 4 mm in diameter and ∼3 mm deep, fabricated from the silicone elastomer, Sylgard (Dow Corning, Midland, MI). Oocytes were exposed to toxin by applying 3 μL of toxin at 10 times its final concentration with a pipettor and manually stirring the bath for a few seconds by gently aspirating and expelling a few microlitres of the bath fluid several times with the pipettor (Wilson et al., 2011a). All toxin exposures were conducted in a static bath to conserve toxin. Toxins were washed out by continuous perfusion with ND96, at an initial rate of 1.5 mL·min⁻¹ for 20 s, followed by a steady rate of 0.5 mL·min⁻¹ (Zhang et al., 2009).

Data analysis

Conductance values were calculated with the formula g_Na = I_Na/(V_step − V_rev), where g_Na is the conductance, I_Na is the peak current amplitude in response to the potential step, V_step is the test potential and V_rev is the reversal potential estimated by extrapolation of the linear part of the I–V curve at positive V_step values, which yielded V_rev values near 50 mV. Normalized activation and inactivation curves were fit to the Boltzmann equation of the form Y = 1/(1 + exp[(V_step − V_1/2)/k]), where Y is the normalized g_Na or I_Na, V_step is the test pulse (for activation curves, stepped in 5 mV increments) or the 500 ms conditioning prepulse (for inactivation curves, stepped in 10 mV increments and immediately preceded the test pulse to −10 mV), V_1/2 is the voltage at half-maximal activation or inactivation and k is the slope factor. Fits of activation and inactivation curves to the Boltzman equation were obtained with Prism software (GraphPad Software Inc., San Diego, CA, USA).

Fitting of time course data to a single-exponential function was done with home-made software written with LabVIEW (e.g. to obtain rates of fast inactivation from the falling phases of current traces).

The interaction of toxin with channel was assumed to be that of simple bimolecular reaction whose kinetics are described by the following equation: k_obs = k_on[toxin] + k_off, with kinetic constants determined as previously described (West et al., 2002). Briefly, the time course of peak I_Na was plotted before, during and after exposure to toxin. The rate constant for the recovery from block, k_off, was determined by fitting the toxin-washout curve to a single-exponential function. However, when recovery from block was very slow (less than 50% recovery after 20 min), k_off was estimated from the level of recovery observed after 20 min of washing and assuming recovery followed a single-exponential time course; these involved k_off values <0.035·min⁻¹ (Zhang et al., 2009; Wilson et al., 2011a). Values of k_off are presented as the mean obtained with n ≥ 9 oocytes. Times longer than 20 min were not used to avoid error due to drift in baseline.

The onset of block was fit to a single-exponential function to yield the observed rate constant, k_obs. Values of k_obs were determined for at least three toxin concentrations (where each concentration was tested with at least three different oocytes) and plotted as a function of [toxin], the slope of the linear regression fit yielded k_on. In principle, the Y-intercept of this plot should yield k_off, but we chose to calculate k_off directly from toxin-washout curves (see above) to avoid extrapolation errors. The dissociation constant, K_d, was calculated from the ratio k_off/k_on. In instances where the level of block achieved a steady state within the experimental time frame of ∼20 min, steady-state dose–response curves were fit to the equation: % block = 100% × (1/(1 + (IC₅₀/[toxin]))), and IC₅₀ values were obtained using either Prism software or KaleidaGraph (Synergy Software, Reading, PA). When a toxin blocked very poorly (i.e. less than half-block was achieved at the highest toxin concentration tested), K_d was estimated from the level of block (% block) achieved at the highest [toxin] tested by use of the Langmuir adsoption isotherm: % block = 100% × (1/(1 + (K_d/[toxin]))).

The tests of co-expression with each the four Na_Vβ subunits yielded a large set of kinetic data, which was condensed to Δk values as follows. The relative change in a kinetic rate constant (either k_on or k_off) induced by co-expression of a Na_Vβ subunit, Δk, was the ratio A × (k⁺/k^–), where k⁺ and k^– are the respective rates with and without Na_Vβ subunit co-expression, and A = 1 or −1 when Na_Vβ subunit co-expression increased k or decreased k respectively. Thus, a positive Δk indicates the factor by which Na_Vβ subunit co-expression increased the rate constant, and a negative Δk indicates the factor by which Na_Vβ subunit co-expression decreased the rate constant.

Data are represented as mean ± SE. Statistical comparisons were performed by two-tailed unpaired t-tests, except for k_on values, where analysis of covariance was performed with Prism software.

Results

All VGSCs examined in this report were those exogenously expressed in oocytes. The intrinsic biophysical properties of α-subunits are altered by co-expression with the various Na_Vβ subunits, and we will first consider this aspect of Na_Vβ subunit co-expression before presenting results regarding the influence Na_Vβ subunit co-expression exerted on the pharmacological properties of VGSCs.

Na_Vβ subunits and the biophysical properties of sodium currents in oocytes exogenously expressing Na_V1.7

Rat Na_Vβ1 through β4 were individually co-expressed with rat Na_V1.7 in oocytes, which were two-electrode voltage clamped to measure voltage-gated sodium currents (I_Na) as described in Methods. Figure 2A shows representative current traces, and the effects of Na_Vβ subunit co-expression on the voltage dependence of activation and inactivation are plotted in Figure 2B. The biophysical parameters are quantified in Table 1, which shows that at least two of the five parameters were significantly changed by co-expression of each of Na_Vβ2 and β4, whereas all five parameters were significantly changed by co-expression of each of Na_Vβ1 and β3. The effects of the latter largely mirrored each other; that is, both Na_Vβ1 and β3 co-expression decreased the time constant of fast inactivation three- to fourfold and shifted the V_1/2 of activation and inactivation by about −10 and −5 mV respectively (Table 1). The effects of co-expression of Na_Vβ1 with Na_V1.7 in our hands are qualitatively consistent with those reported by Chahine's laboratory (Vijayaragavan et al., 2001). Effects of co-expression of the Na_Vβ subunits, particularly the increase in the rate of fast inactivation by Na_Vβ1 and β3, but not by Na_Vβ2 and β4, were also evident with the other three α-subunits, Na_V1.1, 1.2 and 1.6, examined in this report (see Supporting Information Table S1).

Figure 2.

Sodium current traces and activation and inactivation curves of Na_V1.7 without and with co-expression of Na_Vβ1, β2, β3 or β4. X. laevis oocytes were voltage clamped as described in Methods. (A) Representative sodium current traces in response to activation steps, between −50 and 50 mV in 5 mV increments, from a holding potential of −80 mV. (B) activation curves (left, where V_m represents the V_step of the test pulse) and inactivation curves (right, where V_m represents the V_step of the conditioning prepulse) acquired and plotted as described in Methods. Data points represent mean ± SE, with n ≥ 6 oocytes. Solid curves are fits of the data to the Boltzmann equation (see Methods), parameters of which are presented in Table 1.

Table 1.

Activation and inactivation parameters and time constants of fast inactivation of Na_V1.7 without and with Na_Vβ-subunit co-expression

	Activationa		Inactivationb		Inactivationc
NaV1.7	V1/2 (mV)	k (mV)	V1/2 (mV)	k (mV)	τ (ms)
Alone	−10.5 ± 0.3	7.2 ± 0.3	−59.4 ± 1.1	−14.4 ± 1.0	6.1 ± 0.6
+β1	−20.0 ± 0.3*	4.8 ± 0.3*	−64.7 ± 0.4*	−8.2 ± 0.3*	2.1 ± 0.2*
+β2	−9.9 ± 0.3	5.2 ± 0.2*	−60.2 ± 0.9	−10.9 ± 0.8*	6.0 ± 0.3
+β3	−19.1 ± 0.2*	4.8 ± 0.2*	−66.1 ± 0.4*	−7.2 ± 0.4*	1.5 ± 0.2*
+β4	−23.2 ± 0.2*	5.3 ± 0.2*	−59.5 ± 0.6	−11.1 ± 0.5*	6.2 ± 0.3

^aData from Figure 2B, left.

^bData from Figure 2B, right.

^cTime constant of fast inactivation from falling phase of I_Na, in response to voltage step to −10 mV (see e.g. Figure 2A), fit to a single-exponential function (n ≥ 6 oocytes).

^*Statistically different from Na_V1.7 expressed alone (P < 0.05).

Na_Vβ subunit co-expression alters the kinetics of block by μ-conotoxins

The effects of co-expression of Na_Vβ1, β2, β3 or β4 on the block of Na_V1.7 by μ-SmIIIA are illustrated in Figures 3 and 4. Representative sodium current (I_Na) traces are shown in Figure 3A, and representative time courses of block and recovery of from block of peak I_Na are shown in Figure 3B and C respectively. Co-expression of Na_Vβ1 and β3 clearly accelerated the block of Na_V1.7 by μ-SmIIIA, whereas co-expression of Na_Vβ2 and β4 decelerated the block (Figure 3B). Co-expression of Na_Vβ2 and β4 decreased the rate of dissociation of μ-SmIIIA from Na_V1.7, while co-expression of Na_Vβ1 and β3 had minimal effects (Figure 3C).

Figure 3.

Effects of co-expression of Na_Vβ1, β2, β3, or β4 on the kinetics of block of Na_V1.7 by 10 μM μ-SmIIIA. I_Na of voltage-clamped oocytes expressing Na_V1.7 ± Na_Vβ co-expression was measured as described in Methods. Representative I_Na traces (A), time course of block upon exposure to peptide (B) and time course of recovery following peptide washout (C). In panel A, each set of three traces is from a different oocyte: the largest trace represents the control response obtained before exposure to peptide; the smallest trace corresponds to the last response obtained in the presence of peptide in panel B; and the middle trace is the last response obtained during peptide washout in panel C. Each curve in panels B and C is from a different oocyte, where a given symbol represents the co-expression with the same Na_Vβ-subunit in both panels B and C. The observed rate constant of block (k_obs) was obtained by fitting curves, such as those in panel B, each to a single exponential; the slope of the plot of k_obs as a function of peptide concentration (see Figure 4A) yielded the on rate constant, k_on. Values of k_off were obtained by measuring the level of recovery at the end of a 20 min wash (e.g. panel C) and assuming recovery followed a single-exponential time course (or, when >50% recovery was achieved within 20 min, by actually fitting the washout curve to a single exponential, see Methods); resulting k_off values are provided in Table 2.

On-rate constants, k_on, were obtained from plots such as those in Figure 3B by fitting the onset of block to a single-exponential function to obtain the observed rate constant, k_obs. Plots of k_obs versus toxin concentration yielded linear curves (Figure 4A), the slopes of which provided k_on values (see Methods) that are listed in Table 2.

Table 2.

Influence of the co-expression of rat Na_Vβ1, β2, β3 or β4 on the of block of rat Na_V1.1, 1.2, 1.6 or 1.7 by μ-TIIIA, μ-PIIIA or μ-SmIIIA^a

	μ-SmIIIA			μ-PIIIA			μ-TIIIA
	kon (μM·min)−1	koff (min−1)	Kd (μM)	kon (μM·min)−1	koff (min−1)	Kd or IC50b (μM)	kon (μM·min)−1	koff (min−1)	Kd or IC50b (μM)
NaV1.1	2.4 ± 0.1	0.009 ± 0.001	0.0038 ± 0.0004	1.0 ± 0.1	0.053 ± 0.003	0.053 ± 0.004	0.39 ± 0.03	1.10 ± 0.22	0.90 ± 0.08b
+β1	7.1 ± 1.3	0.017 ± 0.003	0.0024 ± 0.0006	6.1 ± 0.8	0.084 ± 0.006	0.014 ± 0.002	0.49 ± 0.04	0.78 ± 0.14	0.71 ± 0.07b
+β2	0.030 ± 0.002	0.0024 ± 0.0003	0.07 ± 0.01	0.044 ± 0.002	0.006 ± 0.001	0.14 ± 0.02	0.35 ± 0.03	1.01 ± 0.08	1.7 ± 0.1b
+β3	4.4 ± 0.5	0.010 ± 0.001	0.0023 ± 0.0003	5.20 ± 0.47	0.09 ± 0.01	0.017 ± 0.003	1.96 ± 0.06	0.84 ± 0.09	0.48 ± 0.05b
+β4	0.010 ± 0.002	0.0027 ± 0.0006	0.30 ± 0.08	0.019 ± 0.002	0.007 ± 0.002	0.37 ± 0.11	0.14 ± 0.04	0.47 ± 0.05	1.66 ± 0.16b
NaV1.2	1.5 ± 0.1	0.0017 ± 0.0004	0.0013 ± 0.0003	0.48 ± 0.04	0.24 ± 0.02	0.62 ± 0.04b	0.44 ± 0.02	0.020 ± 0.002	0.045 ± 0.005
+β1	3.5 ± 0.4	0.0028 ± 0.0007	0.0009 ± 0.0002	1.82 ± 0.28	0.16 ± 0.01	0.24 ± 0.03b	0.41 ± 0.07	0.016 ± 0.002	0.039 ± 0.008
+β2	0.61 ± 0.06	0.0016 ± 0.0002	0.0030 ± 0.0005	0.21 ± 0.02	0.14 ± 0.02	0.65 ± 0.05b	0.49 ± 0.018	0.027 ± 0.003	0.055 ± 0.006
+β3	3.2 ± 0.2	0.0025 ± 0.001	0.0009 ± 0.0003	1.1 ± 0.3	0.20 ± 0.03	0.22 ± 0.02b	0.68 ± 0.05	0.009 ± 0.001	0.015 ± 0.002
+β4	0.40 ± 0.04	0.0030 ± 0.0001	0.0075 ± 0.0008	0.17 ± 0.03	0.14 ± 0.01	1.10 ± 0.05b	0.21 ± 0.014	0.014 ± 0.002	0.067 ± 0.010
NaV1.6c	1.70 ± 0.18	0.12 ± 0.02	0.069 ± 0.013	0.82 ± 0.29	0.067 ± 0.012	0.081 ± 0.032
+β1	2.38 ± 0.05	0.11 ± 0.01	0.046 ± 0.003	7.77 ± 0.45	0.039 ± 0.006	0.005 ± 0.001
+β2	0.05 ± 0.00	0.035 ± 0.003	0.75 ± 0.06	0.19 ± 0.04	0.046 ± 0.006	0.243 ± 0.058
+β3	1.87 ± 0.34	0.11 ± 0.01	0.059 ± 0.013	6.59 ± 0.47	0.062 ± 0.010	0.009 ± 0.002
+β4	0.030 ± 0.007	0.014 ± 0.003	0.40 ± 0.12	0.18 ± 0.02	0.168 ± 0.015	0.951 ± 0.151
NaV1.7d	0.12 ± 0.01	0.031 ± 0.005	0.26 ± 0.05
+β1	0.27 ± 0.05	0.034 ± 0.002	0.13 ± 0.02
+β2	0.010 ± 0.001	0.015 ± 0.002	1.50 ± 0.22
+β3	0.252 ± 0.017	0.027 ± 0.001	0.11 ± 0.01
+β4	0.0063 ± 0.0002	0.0067 ± 0.0004	1.17 ± 0.08
+β112	0.27 ± 0.02	0.027 ± 0.002	0.10 ± 0.01
+β211	0.015 ± 0.001	0.018 ± 0.003	1.20 ± 0.22
+β1+β2	0.047 ± 0.010	0.018 ± 0.003	0.38 ± 0.10
+β1+β4	0.011 ± 0.001	0.0056 ± 0.0004	0.55 ± 0.08
+β3+β2	0.056 ± 0.008	0.011 ± 0.001	0.20 ± 0.03
+β3+β4	0.018 ± 0.001	0.0064 ± 0.0004	0.35 ± 0.03

^aValues are mean ± SE (n ≥ 9 oocytes) and were obtained as described in Methods (see also Figure 3); values for Na_V1.1, 1.2 and 1.7, all without Na_Vβ-subunit co-expression, are from (Wilson et al., 2011a).

^bValue is K_d, but if it has a superscript ‘b’, then it is IC₅₀ (see Methods).

^cNa_V1.6, regardless of Na_Vβ-subunit co-expression, was poorly blocked by μ-TIIIA – at 100 μM, the highest concentration tested, ≲ 30% block was observed in all cases, from which a minimum K_d >200 μM was estimated (see Methods); furthermore, the k_obs and k_off were too large (>2·min⁻¹) to accurately measure, so corresponding area in table is greyed out.

^dNa_V1.7, regardless of Na_Vβ-subunit co-expressed, was essentially insensitive to μ-PIIIA and μ-TIIIA – at 30 μM, the highest concentration tested, each peptide blocked less than 5%, which indicates a minimum K_d > 570 μM, so corresponding areas are greyed out. Plots of k_obs versus [μ-conotoxin], the slopes of which provided k_on values listed here, are illustrated in Figure 4A and Supporting Information Figure S1.

Experiments such as those described in Figures 3 and 4 were also performed for Na_V1.1, 1.2, 1.6 and 1.7 with μ-conotoxins TIIIA, μ-PIIIA as well as μ-SmIIIA. These results are summarized in Table 2. Note that all three toxins blocked Na_V1.1 and 1.2, while Na_V1.6 was blocked by μ-SmIIIA and μ-PIIIA but hardly at all by μ-TIIIA; finally, Na_V1.7 was blocked only by μ-SmIIIA. The estimated lower-limit K_d values of the impotent blockers are given in the footnotes of Table 2.

A conotoxin that blocks Na_V1.7 much like μ-SmIIIA (but more slowly) is μ-KIIIA (Wilson et al., 2011a), so this conopeptide's block of Na_V1.7 was also examined for reasons mentioned in the Introduction, and the results are in Table 3.

Table 3.

Influence of the co-expression of Na_Vβ1, β2, β3 or β4 on the kinetics of block of Na_V1.7 by μ-conotoxin KIIIIA^a

	kon (μM·min)−1	koff (min−1)	Kd (μM)
NaV1.7	0.024 ± 0.002	0.007 ± 0.001	0.292 ± 0.052
+β1	0.041 ± 0.001*	0.010 ± 0.001*	0.244 ± 0.025
+β2	0.0053 ± 0.0007*	0.0073 ± 0.0011	1.38 ± 0.03
+β3	0.031 ± 0.004	0.013 ± 0.001*	0.42 ± 0.06
+β4	0.0030 ± 0.0002*	0.0027 ± 0.0007*	0.93 ± 0.24

^aValues are mean ± SE (n ≥ 9 oocytes). Values for Na_V1.7 without any Na_Vβ-subunit co-expression are from Wilson et al., (2011a).

^*Statistically different than Na_V1.7 without Na_Vβ-subunit co-expression (P < 0.05). Plots of k_obs versus [μ-KIIIA], the slopes of which yielded the k_on values listed here, are shown in the lower-right corner of Supporting Information Figure S1.

Co-expression of two chimeras, Na_Vβ112 and Na_Vβ211, constructed from different parts of Na_Vβ1 and Na_Vβ2, and their effects on μ-SmIIIA's block of Na_V1.7

These Na_Vβ chimera experiments followed the footsteps of others (e.g. Makita et al., 1996; Zimmer and Benndorf, 2002). To determine which portions of the Na_Vβ subunit (i.e. extracellular, intracellular or transmembrane) were responsible for the subunit's effects, two chimeras of Na_Vβ1 and β2 were constructed and tested: the Na_Vβ112 chimera consisted of Na_Vβ1 with its intracellular portion replaced by that of Na_Vβ2, while the Na_Vβ211 chimera consisted of Na_Vβ1 with its extracellular portion replaced by that of Na_Vβ2 (see Figure 4B). Plotted in Figure 4A are data displaying the consequences of the co-expression of the two chimeras on the susceptibility of Na_V1.7 to μ-SmIIIA; the curve representing Na_Vβ112 lies essentially superimposed on those of Na_Vβ1 and β3, while the Na_Vβ211 curve lies near that of Na_Vβ2. The quantified kinetic constants are presented near the bottom of Table 2, which shows that the k_on and k_off values with Na_Vβ112 co-expression are close to the corresponding rate constants with Na_Vβ1 co-expression; likewise, each rate constant with Na_Vβ211 co-expression is close to that corresponding to Na_Vβ2 co-expression. These results suggest that the extracellular portion of the Na_Vβ subunit is largely responsible for the β-subunit's ability to modulate the susceptibility of Na_V1.7 to μ-SmIIIA.

Binary combinations of Na_Vβ subunits co-expressed with Na_V1.7: effects on μ-SmIIIA's kinetics

Thus far, we've only considered unary co-expression of Na_Vβ subunits; however, an α-subunit can associate with a binary combination of Na_Vβ subunits [e.g. β1 and β2 (Hartshorne and Catterall, 1984), or one non-covalently (β1 or β3) and one covalently (β2 or β4) (Patino and Isom, 2010)] to form a ternary complex. We co-expressed Na_Vβ subunits in four binary combinations of Na_Vβ subunits (+β1+β2, +β1+β4, +β3+β2 and +β3+β4) with Na_V1.7, and the block by μ-SmIIIA of each combination was assessed. The unary k_on value of a given β-subunit of a combination differed from that of its partner by at least an order of magnitude (Table 2); nevertheless, the observed rate of block by μ-SmIIIA of the I_Na of oocytes expressing each of the four combinations could be fit by single-exponential functions (not shown), suggesting that in each case a relatively homogeneous population of channels was being expressed. This, in turn, suggests that essentially all the functional channels expressed by given oocyte were likely a ternary complex possessing the same pair of Na_Vβ subunits. The constants for the block of these channels by μ-SmIIIA are presented near the bottom of Table 2 (see also Figure 4A).

Effects of Na_Vβ-subunit co-expression on TTX and STX block of Na_V1.1 and 1.7

In view of the relatively large decreases in k_on produced by co-expression of Na_Vβ2 and β4 with Na_V1.1 and 1.7 (Table 2) (ranging from >5-fold to 240-fold for at least two μ-conopeptides; see Figure 5 below), we examined whether the block of Na_V1.1 and 1.7 by TTX and STX was also affected by Na_Vβ subunit co-expression. The results are presented in Table 4 and summarized as follows. (i) The k_on values of TTX for both Na_V1.1 and 1.7 were essentially the same. (ii) The k_off of TTX for Na_V1.1 was about twice that for Na_V1.7 and presumably accounts for the twofold difference in the IC₅₀ values of TTX for these two Na_V1 isoforms. (iii) The k_off values of STX for Na_V1.1 and 1.7 were essentially the same. (iv) The k_on of STX for Na_V1.1 was larger than that for Na_V1.7 and presumably largely accounts for the difference in the IC₅₀ values of STX for the two Na_V1 isoforms. (v) Overall, Na_Vβ subunit co-expression had no statistically significant effect on the binding properties of TTX and STX, except: (i) Na_Vβ3 co-expression slightly elevated the k_on of TTX for Na_V1.1 (k_on was altered by a factor of 1.25); and (ii) Na_Vβ2 co-expression slightly reduced the k_on of TTX for Na_V1.7 (k_on was altered by a factor of 0.8). Thus, unlike the binding of μ-conotoxins, the binding of STX and TTX to the channel's pore was only minimally affected by co-expression of the channel with Na_Vβ subunits.

Figure 5.

Matrix of plots summarizing the changes in rate constant (Δk) of block by μ-SmIIIA, μ-PIIIA or μ-TIIIA of Na_V1.1, 1.2, 1.6 or 1.7 induced by co-expression with Na_Vβ1, β3, β2 or β4. μ-PIIIA and μ-TIIIA don't block Na_V1.7, and μ-TIIIA blocks Na_V1.6 very poorly, so no results are shown for those combinations; instead, lower right corner shows results for the block by μ-KIIIA of Na_V1.7. Bars represent values of Δk_on or Δk_off (mean ± SE). The Y-axis shows the Δk value, where, as described in Methods, an upward (positive-going) bar indicates the factor by which Na_Vβ co-expression increased k, and a downward (negative-going) bar indicates the factor by which Na_Vβ co-expression decreased k. Note, the scale for both directions is the same in a given plot except the first (where the downward bars are shown with a relatively compressed Y-axis scale and a break in the plot was necessary to accommodate the very large decrease in k_on induced by Na_Vβ4 co-expression (240 ± 49.5, statistically different from Na_V1.1 expressed alone, P < 0.0001). Values of k_on and k_off in Tables 2 and 3 were used to calculate Δk values in these plots. *Statistically different from α-subunit expressed alone (P < 0.05).

Table 4.

Influence of co-expression of Na_Vβ1, β2, β3 or β4 on the block of Na_V1.1 and Na_V1.7 by TTX or STX^a

	TTX			STX
	kon (μM·min)−1	koff (min−1)	IC50 (nM)	kon (μM·min)−1	koff (min−1)	IC50 (nM)
NaV1.1	49.7 ± 4.2	1.17 ± 0.06	13.7 ± 0.6	351.7 ± 27.2	1.50 ± 0.07	2.11 ± 0.06
NaV1.1+β1	55.1 ± 7.1	1.18 ± 0.09	13.7 ± 0.6	358.1 ± 22.1	1.39 ± 0.05	2.10 ± 0.06
NaV1.1+β2	49.1 ± 7.1	1.40 ± 0.08	13.1 ± 0.5	372.0 ± 21.0	1.38 ± 0.07	2.2 ± 0.1
NaV1.1+β3	62.1 ± 1.3*	1.42 ± 0.05	14.0 ± 0.4	320.5 ± 19.2	1.40 ± 0.02	2.26 ± 0.05
NaV1.1+β4	53.5 ± 9.6	1.42 ± 0.13	13.4 ± 0.4	433.9 ± 53.5	1.38 ± 0.05	1.99 ± 0.08
NaV1.7	49.6 ± 3.7	0.46 ± 0.04	7.5 ± 0.4	266.8 ± 33.4	1.66 ± 0.18	5.1 ± 0.4
NaV1.7+β1	56.6 ± 1.1	0.46 ± 0.04	6.3 ± 0.4	251.0 ± 21.5	1.51 ± 0.07	5.2 ± 0.2
NaV1.7+β2	40.0 ± 1.3*	0.38 ± 0.02	7.0 ± 0.3	206.3 ± 9.8	1.42 ± 0.05	4.3 ± 0.2
NaV1.7+β3	47.5 ± 1.0	0.42 ± 0.02	6.8 ± 0.3	236.1 ± 25.7	1.47 ± 0.03	5.1 ± 0.3
NaV1.7+β4	42.8 ± 2.3	0.44 ± 0.03	7.0 ± 0.4	241.9 ± 40.4	1.38 ± 0.06	4.5 ± 0.3

^aValues are mean ± SE (n ≥ 9 oocytes).

^*Statistically different than corresponding Na_V1-subunit expressed without any Na_Vβ-subunit (P < 0.03). Sample current traces of the block by 10 μM TTX, as well as sample time courses for the bock by10 μM TTX and its washout, are illustrated in Supporting Information Figure S2.

Discussion and conclusions

This report reveals a new way to functionally characterized the interaction of β- with α-subunits of VGSCs. Previously, such interactions were assessed almost exclusively by analyzing how Na_Vβ subunit co-expression modulated the gating of the channel (i.e. the biophysical characteristics and parameters such those as illustrated in Figure 2 and listed Table 1). The ensuing figures and tables in this report clearly demonstrate that β-subunit co-expression in oocytes can alter an α-subunit's susceptibility to μ-conotoxins, but much less so to TTX or STX. We discuss our results here under two headings: (i) consequences of Na_Vβ subunit co-expression on the μ-conotoxin susceptibility of VGSCs and (ii) prospects of using μ-conotoxins to identify the Na_Vβ subunit composition of endogenously expressed VGSCs.

(i) Consequences of Na_Vβ subunit co-expression on a channel's interaction with μ-conotoxin

To help distill the large amount of kinetic data presented in Tables 2 and 3, the changes induced by Na_Vβ subunit co-expression on the value of a kinetic constant, Δk, are plotted in Figure 5. The derivation of Δk is given in Methods and outlined in the legend to Figure 5. Thus, Figure 5 represents a matrix of plots summarizing the changes induced by co-expression of each of the four Na_Vβ subunits on the kinetics of block of the various Na_V1 isoforms by the four μ-conotoxins. For the most part, k_on was modestly increased by co-expression of β1 or β3 (e.g. <3-fold with μ-SmIIIA on all Na_V1 isoforms; maximum was ∼9-fold, which was achieved with μ-PIIIA on Na_V1.6). In contrast, k_on could be markedly decreased by co-expression of β2 or β4 (minimally twofold in most instances and >10-fold in four instances: Na_V1.1 with μ-SmIIIA and μ-PIIIA, and both Na_V1.6 and 1.7 with μ-SmIIIA). Alterations in k_off were mostly modest (≲4-fold in most instances with the exception of an ∼8-fold decrease observed in three instances: μ-PIIIA on Na_V1.1 with Na_Vβ2 or β4 and μ-SmIIIA on Na_V1.6 with Na_Vβ4).

Thus, Figure 5 shows two robust effects were observed for the most part; namely, Na_Vβ1 and β3 co-expression increased k_on, and Na_Vβ2 and β4 co-expression decreased k_on, the latter quite strikingly in several instances.

Note that each of the four Na_V1 isoforms examined was susceptible to at least two of the tested μ-conotoxins. Only a minority of the μ-conotoxins discovered thus far are able to block Na_V1.7 (Wilson et al., 2011a); and tests with μ-KIIIA, in addition to those with μ-SmIIIA, show that the aforementioned generalizations regarding Na_Vβ subunit co-expression apply to Na_V1.7. Of note is that μ-KIIIA has only 16 amino acid residues, six fewer than the other μ-conopeptides examined in this report (Figure 1); and additional tests with KIIIA and its synthetic derivatives (e.g. Zhang et al., 2010b) could reveal what factors, such as steric ones, play a role in the modulatory effect of Na_Vβ subunit co-expression on μ-conotoxin activity.

Binary co-expression of Na_Vβ subunits

The block by μ-SmIIIA of Na_V1.7 was also examined in the context of binary co-expression of Na_Vβ subunits. Table 5 recasts the binary co-expression data in Table 2 under the assumption that in a ternary VGSC complex (comprised of one α-subunit and two β-subunits), one of the β-subunits is either Na_Vβ1 or β3, and the other either Na_Vβ2 or β4. The Δk values for unary Na_Vβ co-expression relative to Na_V1.7 alone are shown in the first row of each triplet of rows in Table 5; note that these data are also plotted in the lower left graph in Figure 5. The Δk values for binary relative to unary co-expression are in the remaining rows of Table 5. Note that within each triplet of rows, the respective Δk values are similar (i.e. within about a factor of three of each other). It would appear that the relative changes induced by a given β-subunit's co-expression on k_on (a decrease in the case of β2 and β4, and an increase in the case of β1 and β3) and on k_off (a decrease in the case of β2 and β4, and minimal change in the case of β1 and β3) are not markedly disturbed by the co-expression of an additional β-subunit.

Table 5.

Block of Na_V1.7 by μSmIIIA: Effects of co-expression of binary combinations of Na_Vβ-subunits relative to unary (or no) co-expression of Na_Vβ-subunits^a

β-subunit(s) co-expressedb	Reference NaV1.7c	Δkond	Δkoffd
+β2	alone	−12.0 ± 1.4	−2.1 ± 0.4
+β2+β1	+β1	−5.7 ± 1.6	−1.9 ± 0.3
+β2+β3	+β3	−4.5 ± 0.7	−2.5 ± 0.3
+β4	alone	−20.0 ± 2.1	−4.4 ± 0.8
+β4+β1	+β1	−24.5 ± 5.1	−5.7 ± 0.5
+β4+β3	+β3	−13.9 ± 1.2	−4.5 ± 0.4
+β1	alone	2.3 ± 0.5	1.1 ± 0.2
+β1+β2	+β2	4.7 ± 1.0	1.2 ± 0.3
+β1+β4	+β4	1.8 ± 0.2	−1.2 ± 0.1
+β3	alone	2.1 ± 0.3	−1.1 ± 0.2
+β3+β2	+β2	5.6 ± 0.9	−1.4 ± 0.2
+β3+β4	+β4	3.0 ± 0.2	−1.2 ± 0.1

^aOriginal data are in Table 2.

^bTerminology as in first column of Table 2 for Na_V1.7.

^cChannel used as reference to calculate Δk values; e.g. Δk values with ‘alone’ as reference correspond to those of Na_V1.7 alone (first row in each triplet of rows), which are also plotted in lower-left graph in Figure 5.

^dMagnitude and polarity of a value were obtained as described in Methods (and Figure 5 legend) except for cases with binary β-subunit co-expression, where the reference was Na_V1.7 co-expressed with the β-subunit indicated in second column.

Na_Vβ1 : β2 chimeras

The β112 and β211chimeras behaved much like Na_Vβ1 and Na_Vβ2, respectively (Table 2), consistent with the extracellular domain of the molecule being the major factor contributing to its phenotype. This is reminiscent of Na_Vβ1's modulation of the gating kinetics of Na_V1.2 and 1.4, where the extracellular domain of the Na_Vβ1 subunit was found to contain the determinant site (Chen and Cannon, 1995; Makita et al., 1996; McCormick et al., 1999; Zimmer and Benndorf, 2002).

Block by TTX and STX

The results in Table 4 clearly show that, unlike the binding of μ-conotoxins, the binding of STX and TTX to the channel's pore seems largely immune to co-expression of the channel with Na_Vβ-subunits. Likewise, in our previous experiments, the block of Na_V1.8 by STX was only minimally affected by co-expression of any of the four Na_Vβ subunits – the only significant effects were modest decreases in k_off induced by co-expression of Na_Vβ1 and β4 (k_off reduced by 30% and 40% respectively) (Wilson et al., 2011b). These results are consistent with biochemical experiments involving tritiated-STX binding to Na_V1.2 expressed in Chinese hamster cell lines, which showed that the K_d for STX was not affected by co-expression of Na_Vβ1 (Isom et al., 1995b).

Our recent work showed that TTX/STX can co-occupy site 1 with (at least some) μ-conopeptides and suggested that site 1 might be considered a macrosite (Olivera et al., 1991) consisting of two abutting microsites, one very close to the ion-selectivity filter and accessible to TTX or STX (say, ‘site 1a’) and the other a more superficial site occupiable by μ-conopeptide (say, ‘site 1b’) (Zhang et al., 2009; 2010a, b). Thus, with regard to our present results, it appears that the ‘reach’ of Na_Vβ-subunit co-expression extends to site 1b, but not to site 1a.

Site 1 is in the pore loops (or S5–S6 linkers) of the α-subunit (Cestèle and Catterall, 2000), and Na_Vβ1 is close to these loops insofar as the pore loops of the first and fourth domains of the α-subunit contain important determinants that allow β1 to modulate the gating of the α-subunit (Makita et al., 1996; Catterall, 2000). Our results suggest that the other Na_Vβ subunits may likewise be close to site 1(b).

Other considerations

It should be noted that although it is clear that co-expression of Na_Vβ subunits with the various Na_V1 isoforms perturbed the block of VGSCs by μ-conotoxins, we cannot conclude unequivocally that the association of the β- with the α-subunit in the plasma membrane per se was responsible for the perturbation. Other possibilities exist; for example, the β-subunit could influence posttranslational modifications, such as glycosylation, of the α-subunit during its synthesis.

Regarding glycosylation of Na_Vβ-subunits themselves, mutation of N-linked glycosylation sites (for sialic acids) of Na_Vβ1 and β2 affect their ability, to varying degrees, to modulate the gating of α-subunits (such as Na_V1.2, 1.4, 1.5, and 1.7) expressed in CHO cell lines (Johnson et al., 2004; Johnson and Bennett, 2006). It would be interesting to examine whether Na_Vβ-subunit glycosylation plays a role in the β-subunit's ability to modulate μ-conotoxin binding.

(ii) Prospects of using μ-conotoxins to identify the Na_Vβ subunit composition of endogenously expressed VGSCs

An important point to keep in mind in this regard is that the properties of VGSCs produced by different exogenous expression systems can differ; an example close to home is the following. As noted earlier, the rate of fast inactivation of Na_V1.7 co-expressed with either Na_Vβ1 or β3 was faster than that of Na_V1.7 expressed alone (Figure 1 and Table 2; see also Vijayaragavan et al., 2001). However, this result was not observed when HEK293 cells served as the expression system; there, co-expression of any Na_Vβ isoform (Na_Vβ1-β4) had no effect on the kinetics of Na_V1.7 current (Ho et al., 2012). These investigators also noted that their results with HEK293 cells seemed at odds with the behaviour of TTX-sensitive I_Na in small neurons of DRG of mice (presumably mediated at least in part by Na_V1.7), where fast inactivation is more rapid in control than in mutant mice where Na_Vβ2 expression was knocked out (Lopez-Santiago et al., 2006). It is becoming increasingly clear that the background cell type in which VGSCs are expressed, neuron versus non-neuronal cell line or even among neurons themselves, can influence the physiological properties of VGSCs (Cummins et al., 2001; Dib-Hajj et al., 2009b).

The issue at hand is how important a role does background cell type play in the pharmacological properties of VGSCs (as opposed to their biophysical properties), specifically regarding the pharmacology of site 1. It remains to be seen whether the effects of Na_Vβ subunit co-expression we report here for oocytes also apply to neurons. With regard to the latter, experiments seem called for that involve the use of siRNA to knockdown the expression levels of specific Na_Vβ isoforms (Bant and Raman, 2010) or mutant (knockout) mice where specific Na_Vβ subunits are not expressed altogether (Lopez-Santiago et al., 2006).

In the meantime, we are characterizing the μ-conotoxin susceptibility of sodium currents in different cell types of DRG neurons (Zhang et al., 2013), with the expectation that cell type-specific differences that show up might be correlatable with the different levels of transcripts for the various Na_Vβ subunits expressed by the different cell types (e.g. Ho et al., 2012).

Conclusions

Our discovery that Na_Vβ subunit co-expression can affect the affinity of VGSCs for μ-conotoxins provides yet another avenue through which to explore the interaction of μ-conotoxins with site 1, a potentially important drug target for the therapeutic treatment of neurological disorders where VGSCs are implicated, such as epilepsy and neuropathic pain (Catterall, 2012; Waxman, 2012).

Glossary

DRG

dorsal root ganglion

INa

sodium current

μ-KIIIA

μ-conotoxin KIIIA from Conus kinoshitai

μ-PIIIA

μ-conotoxin PIIIA from Conus pururascens

μ-SmIIIA

μ-conotoxin SmIIIA from Conus stercusmuscarum

μ-TIIIA

μ-conotoxin TIIIA from Conus tulipa

NaV1

α-subunit of voltage-gated sodium channel

NaVβ

β-subunit of voltage-gated sodium channel

NaVβ112

chimera of the extracellular and transmembrane domains of NaVβ1 and intracellular domain of NaVβ2

NaVβ211

chimera of the extracellular domain of NaVβ2 and transmembrane and intracellular domains of NaVβ1

STX

saxitoxin

TTX

tetrodotoxin

VGSC

voltage-gated sodium channel

Conflict of interest

BMO is a cofounder of Cognetix, Inc., and GB is a cofounder of NeuroAdjuvants, Inc.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

Figure S1 Matrix of k_obs versus [μ-conotoxin] plots for the block by μ-SmIIIA, μ-PIIIA or μ-TIIIA of Na_V1.1, 1.2, 1.6, or 1.7, each alone or co-expressed with Na_Vβ1, β3, β2 or β4. Also included is the plot for the block by μ-KIIIA of Na_V1.7 ± Na_Vβ-subunits (lower right). Slopes of these plots provided the k_on values for Tables 2 and 3 (and thus the Δk_on values in Figure 5). Each plot is modelled after Figure 4A, and overall arrangement of plots mirrors that of Figure 5 (including plot in lower right corner illustrating the results for the block by μ-KIIIA of Na_V1.7 ± Na_Vβ subunit co-expression). Thus, columns represent different μ-conotoxins, and rows represent different Na_V1 isoforms. Each plot shows five curves: respective Na_V1 alone (‘no β’), Na_V1 + β1 (‘+β1’), Na_V1 + β2 (‘+β2’), Na_V1 + β3 (‘+β3’) and Na_V1 + β4 (‘+β4’). Note, first two plots in the first row and first plot in the third row each have an inset showing curves for ‘no β’, ‘+β1’ and ‘+β3’ (because these three curves are too close together to easily distinguish among them in the parent plot), where the X-axis has been expanded relative to that of the parent plot; the axes of each inset have the same units as those of the parent plot.

Figure S2 Block by TTX of Na_V1.7 without (left column) and with co-expression of Na_Vβ1 (middle column) or Na_Vβ4 (right column). Top row, sample current traces before (control) and during exposure to 10 nM TTX; in each case, the current was blocked by about 60%. Bottom row, representative time courses of block by 10 nM TTX and recovery following TTX washout. Horizontal black bar represents time interval during which TTX was present. Solid curves represent best fits to single-exponential functions, from which values of k_obs and k_off were obtained. Such k_off values from ≥9 oocytes were averaged to yield each k_off value in Table 4. Also, such k_obs values from 9 oocytes were plotted versus [TTX], and the resulting slope yielded each k_on value in Table 4.

Table S1 Time constant of fast inactivation, τ, for Na_V1.1, 1.2 and 1.6 without and with co-expression of Na_Vβ-subunits.^a