Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium

Abstract

KCNQ2 and KCNQ3 potassium-channel subunits can form both homomeric and heteromeric channels; the latter are thought to constitute native ganglionic M channels. We have tried to deduce the stoichiometric contributions of KCNQ2 and KCNQ3 subunits to currents generated by the coexpression of KCNQ2 and KCNQ3 cDNA plasmids in Chinese hamster ovary (CHO) cells, and to native M currents in dissociated rat superior cervical ganglion (SCG) neurons, by comparing the block of these currents produced by tetraethylammonium (TEA) with the block of currents generated by a tandem KCNQ3/2 construct. TEA concentration-inhibition curves against coexpressed KCNQ2 plus KCNQ3 currents, and against native M currents in SCG neurons from 6-week-old [postnatal day 45 (P45)] rats, were indistinguishable from those for the expressed tandem construct, and fully accorded with a 1:1 stoichiometry. Inhibition curves in neurons from younger (P17) rats could be better fitted assuming an additional small proportion of current carried by KCNQ2 homomultimers. Single-cell PCR yielded signals for KCNQ2, KCNQ3, and KCNQ5 mRNAs in all SCG neurons tested from both P17 and P45 rats. Quantitative PCR of whole-ganglion mRNA revealed stable levels of KCNQ2 and KCNQ5 mRNA between P7 and P45, but excess and incrementing levels of KCNQ3 mRNA. Increasing levels of KCNQ3 protein between P17 and P45 were confirmed by immunocytochemistry. We conclude that coexpressed KCNQ2 plus KCNQ3 cDNAs generate channels with 1:1 (KCNQ2:KCNQ3) stoichiometry in CHO cells and that native M channels in SCG neurons adopt the same conformation during development, assisted by the increased expression of KCNQ3 mRNA and protein.

Figure 1.

Similar kinetics of currents generated by expressed KCNQ3/2tandem cDNA (a) and coexpressed KCNQ2 and KCNQ3 cDNAs in CHO cells (b), and native M/KCNQ currents (c) in dissociated P17 rat SCG neurons. A, Typical currents. Cells were held at -20 mV to preactivate currents. Records show deactivation currents induced by 1 sec hyperpolarizing steps in increments of -10 mV at 20 sec intervals. B, Mean fast (filled triangles) and slow (open squares) deactivation time constants (τ) plotted against deactivation potential. Number of cells: 11 (a), 12 (b), and 17 (c). C, Mean instantaneous (open triangles) and steady-state (filled circles) current–voltage relationships. Number of cells as in B.

Figure 2.

TEA inhibition of separately expressed and concatameric KCNQ2/3 currents are similar and in accord with a single-binding-site model, but two binding sites are required to fit M current inhibition curves in sympathetic neurons from P17 rats. A, Representative deactivation currents recorded on stepping for 1 sec from -20 to -50 mV (Fig. 1) in the presence of incremental concentrations of TEA (0.3–30 mm) for KCNQ3/2 tandem construct (a), coexpressed KCNQ2 and KCNQ3 constructs (b), and native M/KCNQ current (c) in P17 rat SCG neuron. B, Concentration dependence of inhibition of a KCNQ3/2 tandem (filled squares; n = 11) and cotransfected KCNQ2 plus KCNQ3 (open diamonds; n = 12) in CHO cells (a), and native M current (b) in SCG neurons (n = 16–17). Values plotted are means ± SE. Smooth lines superimposed on data sets are least-squares fits to unweighted data points, using one-component(dotted line) and two-component(solid line) Hill equations. Equation 1 is the basic equation. For the coexpressed KCNQ2 plus KCNQ3 constructs in Ba, x₀ = 7.09 ± 0.41 mm and p = 0.78 ± 0.04. For the tandem KCNQ3/2 construct in Ba, x₀ = 3.97 ± 0.23 mm and p = 0.86 ± 0.04. For the dotted curve in Bb, the constants were x₀ = 5.95 ± 0.32 mm and p = 0.48 ± 0.046. The two-component fit (Bb, solid line) is given by Equation 2, in which x₀ and x₁ are additionally the half-inhibitory concentrations (0.05 ± 0.013 and 15.15 ± 1.21 mm, respectively) for two channel types present in proportions q and (1 - q), respectively, and q = 0.24 ± 0.016. C, Concentration dependence fitted as in B but for single cells, exemplified by individual CHO cells expressing KCNQ3/2 tandem and KCNQ2 plus KCNQ3 (a) and an individual SCG neuron M-current (b) (symbols as in B).

Figure 3.

TEA inhibition of M currents in SCG neurons isolated from 6-week-old (P45) rats is in accord with a single-binding-site model. Each point shows the mean inhibition (±SEM) in 10 neurons, measured as described for Figure 2. The solid curve is a single-binding-site fit to Equation 1, with x₀ = 6.0 ± 0.3 mm and p = 0.63 ± 0.02. The interrupted line is a fit to the two-binding-site Equation 2, withx₀=0.27±0.13mm,x₁=12.3±3.8mm,andq=0.25± 0.07. The two-site model did not provide a significantly improved fit (compare Fig. 2 Bb).

Figure 4.

Single-cell PCR indicates the presence of mRNA for KCNQ2,KCNQ3, and KCNQ5, but not KCNQ4, in most SCG neurons tested at P17 and P45. Gels show bands from eight cells from P17 rats (A) and seven cells from P45 rats (B) using the primers described in Materials and Methods. Bands generated from the individual KCNQ cDNA plasmids are shown in the lanes marked P in B. No signals were detected in the absence of cDNA templates. M, Marker ladder.

Figure 5.

Quantitative RT-PCR analysis of KCNQ2 (A), KCNQ3 (B), KCNQ4 (C), and KCNQ5 (D) of whole ganglia isolated from rats at ages E18/E19 (black columns), P7 (white columns), P17 (gray columns), and P45 (hatched columns). KCNQ2–5 transcripts were normalized to GADPH levels and data were expressed as percent of GADPH.

Figure 6.

Immunocytochemical staining of P17 and P45 neurons with antibodies to KCNQ2, KCNQ3, and KCNQ5. The left panels of each pair show bright-field images; the right panels show corresponding dark-field fluorescence images. Note that nearly all neurons in the fields stained for KCNQ2 and KCNQ5 at both P17 and P45, and for KCNQ3 at P45, but that staining for KCNQ3 at P17 was sparser and weaker. Scale bar, 20 μm.

Figure 7.

Double staining for KCNQ2 plus KCNQ3 (A—D, I—L) and KCNQ3 plus KCNQ5 (E—H, M—P) at P17 (A—H) and P45 (I—P). A, E, I, M, Bright-field images (BF). D, H, L, P, Electronic overlays of individual pictures. Note that, whereas most neurons show costaining for KCNQ2 and KCNQ3 at P45, many neurons in the field stain more strongly for KCNQ2 than for KCNQ3 at P17. Scale bar, 20 μm.