Fig. 3

Expression of the kcnk4b gene in trunk muscle of developing zebrafish and channel activity of kcnk4b heterologously expressed in HEK-293 cells and in mouse skeletal muscle fibers. (A) Relative quantification of kcnk4b gene by real-time RT-PCR analysis using RNA extracts of 1-wk postfertilization trunks (n = 30 for each experiment), skeletal muscle isolated from 1-mo postfertilization trunks (n = 4 for each experiment), and 1-y old adult fish trunks (n = 1 for each experiment). Three independent experiments were performed. Data were normalized to polr2d. Expression levels were compared to kcnk4b gene expression at 1 wpf, which was arbitrarily set to 1. (B) Confocal image of HEK cells expressing TRAAK-GFP proteins. Arrows indicate the TRAAK-GFP-positive membrane regions on which patch pipettes were sealed. (C) Burst of single-channel outward currents evoked at 0 mV in response to negative pressure applied in a patch pipette sealed on a TRAAK-GFP-positive membrane region of a HEK cell membrane. (D) Recording of single-channel activity elicited by negative pressure in HEK cell-attached patches held at different membrane potentials. (E) Relationship between mean stretch-activated unitary current amplitudes in HEK cell–attached patches and membrane potential (n = 11). (F) Confocal image of a mouse muscle fiber expressing TRAAK-GFP protein after electroporation of kcnk4b-EGFP plasmid. (G) Burst of single-channel outward currents evoked at 0 mV in response to a brief negative pressure applied in a patch pipette sealed on the GFP-positive region of a mouse muscle cell. (H) Recording of single-channel activity elicited by negative pressure in mouse muscle cell–attached patches held at different membrane potentials. (I) Relationship between mean stretch-activated unitary current amplitudes in mouse muscle cell-attached patches and membrane potential (n = 14). For all cell-attached experiments, Tyrode and K+-rich solution were present in the pipette and in the bath, respectively.