[
International C. elegans Meeting,
2001]
CLH-3 is a ClC-2 Cl - channel ortholog expressed in C. elegans oocytes. The channel is activated by oocyte swelling and during meiotic maturation. Activation of CLH-3 in maturing oocytes modulates ovulatory contractions of surrounding gap junction-coupled sheath cells. The CLH-3 protein contains consensus motifs for phosphorylation by cyclin-dependent and MAP kinases, both of which play critical roles in regulating meiotic maturation and other cell cycle processes. Given these observations, we postulated that CLH-3 activation is regulated by protein phosphorylation events. To begin testing this hypothesis, we carried out a series of pharmacological studies. In control, non-maturing oocytes, CLH-3 is inactive (mean whole-cell current at -70 mV = -3.1 +/- 0.3 pA/pF). When oocytes were pretreated with metabolic inhibitors for 15-20 minutes, CLH-3 activity was detected immediately upon obtaining whole-cell access (mean current = -51 +/- 12 pA/pF). Dialysis of oocytes with an ATP-free, metabolic inhibitor-containing pipette solution activated CLH-3 in the absence of swelling. Incubation of oocytes with 100 nM calyculin A, a PP1/PP2A phosphatase inhibitor, inhibited swelling-induced current activation by 88 +/- 4%. However, extracellular incubation and intracellular dialysis of cells for 10 min with 1 m M okadaic acid, a more potent PP2A inhibitor, had no effect on current activation induced by cell swelling. To examine the role of phosphorylation in channel activation during meiotic maturation, we carried out a series of experiments on oocytes maturing in vitro . Shortly before maturation begins, the oocyte nucleus increases in size and migrates to the cell periphery. Oocytes with off-center nuclei undergo maturation within 6.2 +/- 1 min after isolation from the gonad. CLH-3 is constitutively active in maturing oocytes (mean current = -44.4 +/- 6.2 pA/pF). Exposure of maturing oocytes to 100 nM calyculin A completely inhibited maturation-induced channel activation whereas 1 m M okadaic acid had no effect. Taken together, these experimental results demonstrate that serine/threonine dephosphorylation events activate CLH-3. The differential effect of calyculin A versus okadaic acid suggests strongly that protein dephosphorylation is mediated by a PP1-type phosphatase. Almost nothing is known about the signaling pathways that regulate ClC-type Cl - channels. We have begun to use RNA interference in an effort to identify the phosphatases involved in CLH-3 regulation. Analysis of the C. elegans genome revealed the presence of 15 genes that encode PP1-like phosphatases. Recent microarray studies (Hill et al., Science 290:809-812, 2000) have demonstrated that at least four PP1-like phosphatases are expressed in oocytes. In addition, oocytes express two novel phosphatases encoded by C24H11.1 and R13A5.11. RNA interference of CePP1 b and CePP1 g produced an embryonic lethal phenotype, but had no effect on swelling-induced channel activation. Channel activity was normal and no obvious phenotypes were detected with knockdown of C24H11.1 and R13A5.11 expression. In addition to RNA interference, we are also using yeast 2 hybrid analysis, heterologous expression and mutagenesis methods, and biochemical approaches to identify phosphatases and kinases that modulate channel activity. Our long term goal is to identify the signaling pathways and interacting proteins responsible for regulation of CLH-3 and its mammalian ortholog ClC-2.