Fig 2: Expression of the other neuronal KCNQ subunits in the PPN. KCNQ2, KCNQ3, and KCNQ5 staining of coronal PPN sections in wild-type (WT, Kcnq4+/+) (A) and knockout (KO, Kcnq4–/–) (B) mice. KCNQ2 was found in neuron fibers without co-localization in cholinergic neurons. KCNQ3 and KCNQ5 were located in the soma of both cholinergic (yellow arrows) and non-cholinergic neurons. For KCNQ2 and KCNQ5, there was no difference between genotypes. Besides, KCNQ3 in KO animals labeled mostly non-cholinergic (white arrows) neurons. Upper panel: KCNQ subunit (green). Middle panel: ChAT (red). Bottom panel: merged with DAPI. Scale bar, 20 µm.

Fig 3: The presence of the KCNQ4 subunit can be detected in a subpopulation of PPN cholinergic neurons. (A) Actions of the nonspecific KCNQ opener retigabine on the M-current (black: control trace; blue: trace with retigabine). (B) Representative M-current traces under control conditions (black) and with the KCNQ2- and KCNQ4-specific opener ML213 (blue). (C) Representative M-current traces under control conditions (black) and with the KCNQ2- and KCNQ3-specific opener ICA27243 (blue). Dashed lines indicate the holding currents in the control. Dotted lines indicate 0 pA. (D) Statistical comparisons of the currents activated by retigabine (R), ML213 (M), and ICA27243 (I) at –20 mV holding potential (green: outward currents greater than 10 pA; red: currents less than 10 pA; black squares: mean ± SD). With retigabine, the holding current at –20 mV increased from 95.25 ± 11.96 to 140.23 ± 12.24 pA (n = 11, p = 0.0089). With ML213, the increase of the holding current at –20 mV holding potential was 25.61 ± 5.94 pA (from 143.99 ± 28.00 to 169.61 ± 29.66 pA; n = 13, p = 0.269). With ICA27243, the increase in the outward current was 19.00 ± 8.61 pA (from 165.04 ± 23.23 to 174.96 ± 25.78 pA; n = 7, p = 0.389). (E–G) Representative voltage traces obtained with 100 pA depolarizing current injections under the control conditions (black) and with non-selective (retigabine) *p < 0.050. (E), KCNQ2- and KCNQ4-selective (ML213) (F), and KCNQ2- and KCNQ3-selective (ICA27243) (G) openers. Dotted lines indicate 0 mV. (H) Statistical comparison of the changes in AI by openers with different selectivities (green: increase; red: no change or decrease in individual data; black squares: mean ± SD). ***p < 0.001 (significant difference). With retigabine, the AI increased from 0.330 ± 0.035 to 0.755 ± 0.100 (n = 9, p = 0.0005), with ML213, the AI changed from 0.357 ± 0.066 to 0.608 ± 0.131 (n = 10, p = 0.067), and with ICA27243, the AI was 0.392 ± 0.025 in the control and 0.439 ± 0.098 after drug application (n = 10, n.s.).

Fig 4: Immunolocalization of a- and ß-subunits of KCNQ channels in the rat rectal colon. a Fluorescence of KCNQ2 on the luminal membranes of surface cells (arrowheads). b Fluorescence image of ezrin. c Overlay image of a and b. d Overlay image of ezrin and green fluorescence with KCNQ2 antibody pre-absorbed with the control peptide antigen. e Fluorescence of KCNQ4 on the luminal membrane of crypt cells (arrowheads). Fluorescence images of ezrin (f), the overlay (g), and negative control with the pre-absorbed KCNQ4 antibody (h). i Fluorescence of KCNE3 on the luminal membrane of crypt cells (arrowheads). Fluorescence images of ezrin (j), the overlay (k), and negative control with the pre-absorbed KCNE3 antibody (l). Fluorescence of KCNQ1 (m), ezrin (n), and the overlay (o) in crypts. Arrowheads show the luminal membrane of crypt cells. p Negative control with the pre-absorbed KCNQ1 antibody. DAPI was used to stain nuclei (blue). Bars = 20 µm

Fig 5: RT-PCR analysis of a- and ß-subunits of KCNQ channels from the mucosa of rectal colon (RC), distal colon (DC), and ileum (IL). The primers used gave the expected fragment lengths for Kcnq1 (613 bp; a), Kcnq2 (510 bp; b), Kcnq3 (790 bp; c), Kcnq4 (664 bp; d), Kcnq5 (502 bp; e), Kcne1 (281 bp; f), Kcne2 (224 bp; g), Kcne3 (232 bp; h), Kcne4 (476 bp; i), and Kcne5 (228 bp; j). RNA was treated with (+) or without (-) reverse transcriptase (RT). Positive controls obtained from brain (B) or kidney (K). A representative gel for at least three independent experiments is shown. M, molecular mass