Molecular correlates of the M-current in cultured rat hippocampal neurons

Abstract

M-type K(+) currents (I(K(M))) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K(+) channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I(K(M)) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I(K(M)) with an IC(50) of 3.6 +/- 1.5 microM. In 70 % of these cells, TEA fully suppressed I(K(M)) with an IC(50) of 0.7 +/- 0.1 mM. In the remaining cells, TEA maximally reduced I(K(M)) by only 59.7 +/- 5.2 % with an IC(50) of 1.4 +/- 0.3 mM; residual I(K(M)) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I(K(M)) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.

Figure 1. Reverse-transcription PCR analysis from rat pyramidal hippocampal neurons

A, RT-PCR was performed using 0.2 μg of DNase I-treated, total RNA from CA1 or CA3 hippocampal regions isolated from two different 5- to 7-day-old rats. See Methods for intron-spanning primer pairs. Control reactions were performed using plasmids containing cDNA sequences encoding rKCNQ2, rKCNQ3, rKCNQ4 and hKCNQ5 (labelled 2 to 5). -ve, absence of template; M, 1 kb plus ladder (Life Technologies). B, representative single-cell PCRs for KCNQ2-5 and VGLUT1 mRNAs obtained from three single hippocampal neurons (labelled A, B and C). Cells were isolated from the CA1 and CA3 regions of 7-day-old rats and subsequently cultured in vitro for 10 days. Primer pairs used are described in Methods. Control reactions were performed using plasmids containing cDNA sequences encoding rKCNQ2, rKCNQ3, rKCNQ4 and hKCNQ5 (labelled 2 to 5). Hipp, rat hippocampus cDNA; -ve, absence of template; M, 1 kb plus ladder.

Figure 2. Immunodetection of KCNQ channel subunits in hippocampal neurons

Confocal images (10 stacked at 0.5 μm intervals) of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 immunostaining in hippocampal pyramidal neurons cultured in vitro for 10 days. Note the plasma membrane staining for KCNQ2 whereas KCNQ3 and KCNQ5 antibodies appear to label both the cell surface and intracellular components. Inset, staining of CHO cells expressing exogenous KCNQ4 subunits with the anti-KCNQ4 antibody. Scale bar, 10 μm. The greyscale insets show phase-contrast images of the same fields at 40 % magnification.

Figure 3. Pharmacological characterization of I_K(M) in hippocampal pyramidal cells

A and B show currents recorded from two cultured neurons in the absence (Control) and presence of oxotremorine-M (Oxo-M) and linopirdine, respectively. Cells were held at −20 mV and currents evoked by stepping to −50 mV (voltage protocol shown in A). The difference currents were obtained by subtracting the control trace at −50 mV from that in the presence of the respective compounds. The horizontal dashed lines mark the initial baseline holding current. The scale bars shown in B also apply to A. C, average concentration-inhibition curve for linopirdine fitted using eqn (1). Each data point is the mean ±s.e.m. of 3–13 observations. (The slow component of the difference current in A may reflect the hyperpolarization-activated cation current I_Q/I_h, which is enhanced by oxotremorine-M: see Colino & Halliwell, 1993.)

Figure 4. Effects of TEA on I_K(M) recorded in hippocampal pyramidal neurons

A and B show representative examples of currents recorded from two different cells with differential sensitivity to 10 mm TEA. Superimposed are currents in the presence of TEA and co-applied TEA and linopirdine (Linop, 30 μM). The currents were recorded by applying the voltage step shown in A. C, cumulative concentration-inhibition curves for TEA. Squares and circles represent curves for cells in which TEA abolished I_K(M) (n= 7) and cells in which TEA only partially inhibited I_K(M) (n= 3), respectively.