Jerod S. Denton

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons

The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Intracellular H+ regulates the α-subunit of ENaC, the epithelial Na+ channel

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (α, β, γ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of α-, β-, and γ-subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the α-ENaC subunit was examined in planar lipid bilayers. We report that α,β,γ-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited α,β-ENaC, α,γ-ENaC, and α-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the α-ENaC subunit is regulated directly by pHi.

High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1

The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K+ (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.

GCK-3, a Newly Identified Ste20 Kinase, Binds To and Regulates the Activity of a Cell Cycle–dependent ClC Anion Channel

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

The NH2 Terminus of the Epithelial Sodium Channel Contains an Endocytic Motif

An epithelial sodium channel (ENaC) is composed of three homologous subunits: α, β, and γ. To elucidate the function of the cytoplasmic, NH2 terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopusoocytes. Amiloride-sensitive Na+ currents (INa) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH2 terminus of α (Δ2–109), β (Δ2–49), or γ-rENaC (Δ2–53) dramatically reduced INa. A series of progressive, NH2-terminal deletions of α-rENaC were constructed to identify motifs that regulate INa. Deletion of amino acids 2–46 had no effect on INa: however, deletion of amino acids 2–51, 2–55, 2–58, and 2–67 increased INa by ∼4-fold. By contrast, deletion of amino acids 2–79, 2–89, 2–100, and 2–109 eliminated INa. To evaluate the mechanism whereby Δ2–67-α-rENaC increased INa, single channels were evaluated by patch clamp. The single-channel conductance and open probability of α,β,γ-rENaC and Δ2–67-α,β,γ-rENaC were similar. However, the number of active channels in the membrane increased from 6 ± 1 channels per patch with α,β,γ-rENaC to 11 ± 1 channels per patch with Δ2–67-α,β,γ-rENaC. Laser scanning confocal microscopy confirmed that there were more Δ2–67-α,β,γ-rENaC channels in the plasma membrane than α,β,γ-rENaC channels. Deletion of amino acids 2–67 in α-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 ± 0.2 to 3.5 ± 1.1 h. We conclude that the cytoplasmic, NH2 terminus of α-, β-, and γ-rENaC is required for channel activity. The cytoplasmic, NH2 terminus of α-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.

Cell cycle– and swelling-induced activation of a Caenorhabditis elegans ClC channel is mediated by CeGLC-7α/β phosphatases

ClC voltage-gated anion channels have been identified in bacteria, yeast, plants, and animals. The biophysical and structural properties of ClCs have been studied extensively, but relatively little is known about their precise physiological functions. Furthermore, virtually nothing is known about the signaling pathways and molecular mechanisms that regulate channel activity. The nematode Caenorhabditis elegans provides significant experimental advantages for characterizing ion channel function and regulation. We have shown previously that the ClC Cl− channel homologue CLH-3 is expressed in C. elegans oocytes, and that it is activated during meiotic maturation and by cell swelling. We demonstrate here that depletion of intracellular ATP or removal of Mg2+, experimental maneuvers that inhibit kinase function, constitutively activate CLH-3. Maturation- and swelling-induced channel activation are inhibited by type 1 serine/threonine phosphatase inhibitors. RNA interference studies demonstrated that the type 1 protein phosphatases CeGLC-7α and β, both of which play essential regulatory roles in mitotic and meiotic cell cycle events, mediate CLH-3 activation. We have suggested previously that CLH-3 and mammalian ClC-2 are orthologues that play important roles in heterologous cell–cell interactions, intercellular communication, and regulation of cell cycle–dependent physiological processes. Consistent with this hypothesis, we show that heterologously expressed rat ClC-2 is also activated by serine/threonine dephosphorylation, suggesting that the two channels have common regulatory mechanisms.

Development of a Selective Small-Molecule Inhibitor of Kir1.1, the Renal Outer Medullary Potassium Channel

The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2′-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target.

Eliciting Renal Failure in Mosquitoes with a Small- Molecule Inhibitor of Inward-Rectifying Potassium Channels

Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of Aedes aegypti (AeKir1). Injection of VU573 into the hemolymph of adult female mosquitoes (Ae. aegypti) disrupts the production and excretion of urine in a manner consistent with channel block of AeKir1 and renders the mosquitoes incapacitated (flightless or dead) within 24 hours. Moreover, the toxicity of VU573 in mosquitoes (Ae. aegypti) is exacerbated when hemolymph potassium levels are elevated, suggesting that Kir channels are essential for maintenance of whole-animal potassium homeostasis. Our study demonstrates that renal failure is a promising mechanism of action for killing mosquitoes, and motivates the discovery of selective small-molecule inhibitors of mosquito Kir channels for use as insecticides.

Alternative splicing of N- and C-termini of a C. elegans ClC channel alters gating and sensitivity to external Cl- and H+.

CLH‐3 is a meiotic cell cycle‐regulated ClC Cl− channel that is functionally expressed in oocytes of the nematode Caenorhabditis elegans. CLH‐3a and CLH‐3b are alternatively spliced variants that have identical intramembrane regions, but which exhibit striking differences in their N‐ and C‐termini. Structural and functional studies indicate that N‐ and C‐terminal domains modulate ClC channel activity. We therefore postulated that alternative splicing of CLH‐3 would alter channel gating and physiological functions. To begin testing this hypothesis, we characterized the biophysical properties of CLH‐3a and CLH‐3b expressed heterologously in HEK293 cells. CLH‐3a activates more slowly and requires stronger hyperpolarization for activation than CLH‐3b. Depolarizing conditioning voltages dramatically increase CLH‐3a current amplitude and induce a slow inactivation process at hyperpolarized voltages, but have no significant effect on CLH‐3b activity. CLH‐3a also differs significantly in its extracellular Cl− and pH sensitivity compared to CLH‐3b. Immunofluorescence microscopy demonstrated that CLH‐3b is translationally expressed during all stages of oocyte development, and furthermore, the biophysical properties of the native oocyte Cl− current are indistinguishable from those of heterologously expressed CLH‐3b. We conclude that CLH‐3b carries the oocyte Cl− current and that the channel probably functions in nonexcitable cells to depolarize membrane potential and/or mediate net Cl− transport. The unique voltage‐dependent properties of CLH‐3a suggest that the channel may function in muscle cells and neurones to regulate membrane excitability. We suggest that alternative splicing of CLH‐3 N‐ and C‐termini modifies the functional properties of the channel by altering the accessibility and/or function of pore‐associated ion‐binding sites.