Figure 6. GCK-3 regulates CLH-3b activity in vivo. (A)
gck-3 and
clh-3 are coexpressed in C. elegans oocytes and the excretory cell. Left, detection of GCK-3 transcripts in single worm oocytes by RT-PCR. Expected product sizes of amplified GCK-3 cDNA and genomic DNA are 0.8 kb and 3.5 kb, respectively. Transcripts were not detected in samples of oocyte wash buffer. Right, combined confocal differential interference contrast and fluorescence micrographs showing expression of the
gck-3 transcriptional reporter, Pgck-3::GFP, in the H-shaped excretory cell. This GFP expression pattern was observed in two independent lines of transgenic worms. (B) GCK-3 knockdown constitutively activates CLH-3b in meiotically arrested C. elegans oocytes. Whole cell currents measured in GCK-3 RNAi oocytes between -40 and -100 mV were significantly (P 0.004) different form those measured in control GFP RNAi oocytes. Values are means = SEM (n = 8-11). GFP and GCK-3 dsRNA injections were performed in parallel on two separategroups of worms. (C) CLH-3b activation voltages in oocytes isolated from GFP and GCK-3 dsRNA-injected worms. Values are means +/- SEM (n = 8-11). P < 0.0001 compared with oocytes from GFP dsRNA-injected worms. P < 0.0001 compared with nonswollen GCK-3 RNAi oocytes. (D) Relative swelling-induced whole cell current in GFP or GCK-3 RNAi oocytes. Cells were swollen for 5 min. Values are means +/- SEM (n = 5-6). Relative current values were calculated only for test potentials where the current amplitude was measurable.