Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl− cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. Liddle's syndrome is caused by gain-of-function mutations in the β and γ subunits of ENaC, resulting in enhanced Na reabsorption and hypertension. Epidermal growth factor (EGF) causes acute inhibition of Na absorption in collecting duct principal cells via an extracellular signal–regulated kinase (ERK)–dependent mechanism. In experiments with primary cultures of collecting duct cells derived from a mouse model of Liddle's disease (β-ENaC truncation), it was found that EGF inhibited short-circuit current (Isc) by 24 ± 5% in wild-type cells but only by 6 ± 3% in homozygous mutant cells. In order to elucidate the role of specific regions of the β-ENaC C terminus, Madin-Darby canine kidney (MDCK) cell lines that express β-ENaC with mutation of the PY motif (P616L), the ERK phosphorylation site (T613A), and C terminus truncation (R564stop) were created using the Phoenix retroviral system. All three mutants exhibited significant attenuation of the EGF-induced inhibition of sodium current. In MDCK cells with wild-type β-ENaC, EGF-induced inhibition of Isc (<30 min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (Po). At later times (>30 min), EGF-induced inhibition of Isc was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. Our results are consistent with an ERK-mediated decrease in ENaC open probability and enhanced retrieval of sodium channels from the apical membrane.

-Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I sc) by 15-25%, whereas the addition of ATP to the apical bathing solution decreased Isc by 40 -60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I sc in mCT12 monolayers by 46 Ϯ 4% (n ϭ 8) and 22 Ϯ 2% (n ϭ 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 M) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I sc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 M) almost completely blocked the PMA-induced decrease in I sc, but did not alter the EGF-or ATP-induced inhibition of Isc. The DBHQ-mediated decrease in I sc was due to inhibition of basolateral Na ϩ -K ϩ -ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na ϩ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia.mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel AMILORIDE-SENSITIVE Na ϩ transport, mediated by the epithelial Na ϩ channel (ENaC), is an important pathway for Na ϩ absorption by the colon and for Na ϩ retention by the distal nephron (16). A similar mechanism for Na ϩ transport is in the salivary duct, urinary bladder, sweat duct, and airway epithelium, where the process is responsible for the clearance of fetal lung liquid at birth (9, 28) and for control of the composition and/or depth of airway surface liquid in the postnatal lung (4). Defective regulation of ENaC and resultant Na ϩ hyperabsorption by the airway surface epithelium is thought to be an important component of the pathophysiology of cystic fibrosis (5). ENaC loss-of-function mutations (e.g., pseudohypoaldosteronism type I) are known to cause hyponatremia, hyperkalemia, and salt wasting, whereas gain-of-function mutations (e.g., salt-sensitive hypertension, Liddle's syndrome) lead to an increase in ENaC activity and excess Na ϩ reabsorption (20, 31).Corticosteroids, insulin, and vasopressin cause acute and/or chronic stimulation of sodium transport in epithelial cells (45) by several mechanisms, including alterations in ENaC open probability (23), increased synthesis of channel subunits...

Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl(-) cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes.

clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans voltage‐gated ClC Cl‐ channel. CLH‐3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC‐7α and GLC‐7β. GCK‐3 is a Ste20 kinase and homolog of mammalian PASK/SPAK, which regulates cation‐coupled Cl‐ cotransporters. GCK‐3 binds to a 101 amino acid C‐terminal splice insert unique to CLH‐3b. When co‐expressed with CLH‐3b, GCK‐3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Knockdown of GCK‐3 in worm oocytes constitutively activates CLH‐3b. Using mass spectrometry, we identified a serine residue (S747) located within the 101 amino acid splice insert that is phosphorylated in the presence of GCK‐3. S747 and surrounding amino acids conform to the Ste20 kinase phosphorylation motif. Mutation of S747 to alanine prevents GCK‐3 mediated channel inhibition and alterations of channel voltage‐dependent properties. We conclude that phosphorylation of S747 is critical for regulation of CLH‐3b activity. S747 may be phosphorylated directly by GCK‐3 or other downstream kinases.Supported by NIH R01 grant DK51610