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Keith Nehrke

Publications and source records attributed to Keith Nehrke.

At least 19 recordsLinked to original sources
PubMed2006

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis.

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Amino Acid Sequence↗
PubMed2006

Carboxy terminus splice variation alters ClC channel gating and extracellular cysteine reactivity.

CLH-3a and CLH-3b are Caenorhabditis elegans ClC channel splice variants that exhibit striking differences in voltage, Cl(-), and H(+) sensitivity. The major primary structure differences between the channels include a 71 amino acid CLH-3a N-terminal extension and a 270 amino acid extension of the CLH-3b C-terminus. Deletion of the CLH-3a N-terminus or generation of a CLH-3a/b chimera has no effect on channel gating. In contrast, deletion of a 169 amino acid C-terminal CLH-3b splice insert or deletion of the last 11 amino acids of cystathionine-beta-synthase domain 1 gives rise to functional properties identical to those of CLH-3a. Voltage-, Cl(-)-, and H(+)-dependent gating of both channels are lost when their glutamate gates are mutated to alanine. Glutamate gate cysteine mutants exhibit similar degrees of inhibition by MTSET, but the inhibition time constant of CLH-3b is sevenfold greater than that of CLH-3a. Differences in MTSET inhibition are reversed by deletion of the same cytoplasmic C-terminal regions that alter CLH-3b gating. Our results indicate that splice variation of the CLH-3b cytoplasmic C-terminus alters extracellular structure and suggest that differences in the conformation of the outer pore vestibule and associated glutamate gate may account for differences in CLH-3a and CLH-3b gating.

Amino Acid Substitution↗
PubMed2006

Genetic hypercalciuric stone-forming rats.

PURPOSE OF REVIEW: We will describe the pathophysiology of hypercalciuria and the mechanism of the resultant stone formation in a rat model and draw parallels to human hypercalciuria and stone formation. RECENT FINDINGS: Through inbreeding we have established a strain of rats that excrete 8-10 times more urinary calcium than control rats. These genetic hypercalciuric rats absorb more dietary calcium at lower 1,25-dihydroxyvitamin D3 levels. Elevated urinary calcium excretion on a low-calcium diet indicated a defect in renal calcium reabsorption and/or an increase in bone resorption. Bone from hypercalciuric rats released more calcium when exposed to 1,25-dihydroxyvitamin D3. Bisphosphonate significantly reduced urinary calcium excretion in rats fed a low-calcium diet. Clearance studies showed a primary defect in renal calcium reabsorption. The intestine, bone and kidneys of the hypercalciuric rats had increased numbers of vitamin D receptors. When hydroxyproline is added to their diet they form calcium oxalate stones, the most common stone type in humans. Increased numbers of vitamin D receptors may cause hypercalciuria in these rats and humans. SUMMARY: Understanding the mechanism of hypercalciuria and stone formation in this animal model will help clinicians devise effective treatment strategies for preventing recurrent stone formation in humans.

Adsorption↗
PubMed2006

Ste20-type kinases: evolutionarily conserved regulators of ion transport and cell volume.

Ste20 serine/threonine kinases regulate fundamental cellular processes including the cell cycle, apoptosis, and stress responses. Recent studies in Caenorhabditis elegans and mammals demonstrate that Ste20 kinases also function in cell volume sensing and Cl- transport regulation. Yeast Ste20 initiates a shrinkage activated MAPK cascade that regulates organic osmolyte accumulation. Ste20 kinases thus play evolutionarily conserved roles in cellular volume sensing and regulation. They may also function in systemic osmotic homeostasis and to link cell-cycle events with cell volume.

Alanine↗
PubMed2006

Intracellular pH measurements in vivo using green fluorescent protein variants.

Whether by patch-clamp techniques or the use of fluorescent vital dyes, measurements of transepithelial ion flux in mammals are limited by cell accessibility. Furthermore, redundant functions and complex regulatory mechanisms can mask loss-of-function phenotypes through compensatory mechanisms. In this chapter, we present a technique whereby the optically transparent nematode Caenorhabditis elegans, engineered to express a fluorescent pH indicator protein, can be used to study how intracellular pH (pHi) fluctuates in response to environmental and/or experimental challenge. By using a live whole animal model, systemic, and even behavioral relationships to individual cellular pHi can be inferred. In combination with dye loading of excised or cultured cells, this technique also provides a powerful means of contrasting these relationships to biophysical measurements of ion flux.

Animals↗
PubMed2005

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte.

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH(2) and COOH termini. The major differences include a 71-amino acid CLH-3a NH(2)-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl(-) sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bDeltaC, in its native cellular environment. CLH-3bDeltaC exhibits altered voltage-dependent gating as well as pH and Cl(-) sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn(2+), prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bDeltaC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs.

Adenosine Triphosphate↗
PubMed2005

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Animals↗
PubMed2005

The abts and sulp families of anion transporters from Caenorhabditis elegans.

The slc4 and slc26 gene families encode two distinct groups of gene products that transport HCO(3)(-) and other anions in mammalian cells. The SLC4 and SLC26 proteins are important contributors to transepithelial movement of fluids and electrolytes and to cellular pH and volume regulation. Herein we describe the cDNA cloning from the nematode Caenorhabditis elegans of four anion bicarbonate transporter (abts) homologs of slc4 cDNA and eight sulfate permease (sulp) homologs of slc26 cDNA. Analysis of transgenic nematode strains carrying promoter::GFP fusions suggests relatively restricted expression patterns for many of these genes. At least three genes are expressed primarily in the intestine, three are expressed primarily in the excretory cell, and one is expressed in both of these polarized cell types. One of the genes is also expressed exclusively in the myoepithelium-like cells of the pharynx. Many of the sulp gene products localize to the basolateral membrane rather than to the apical membrane. Several ABTS and SULP proteins exhibited anion transport function in Xenopus oocytes. The strongest Cl(-) transporter among these also mediated Cl(-)/HCO(3)(-) exchange. These findings encourage exploitation of the genetic strengths of the nematode model system in the study of the physiological roles of anion transport by the proteins of these two highly conserved gene families.

Amino Acid Sequence↗
PubMed2005

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-7alpha and GLC-7beta. 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.

Animals↗
PubMed2004

Physiological roles of the intermediate conductance, Ca2+-activated potassium channel Kcnn4.

Three broad classes of Ca(2+)-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1-3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K(+) permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K(+) permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland.

Animals↗
PubMed2003

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.

Alternative Splicing↗
PubMed2003

A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.

Na+/H+ exchangers are involved in cell volume regulation, fluid secretion and absorption, and pH homeostasis. NHX-2 is a Caenorhabditis elegans Na+/H+ exchanger expressed exclusively at the apical membrane of intestinal epithelial cells. The inactivation of various intestinal nutrient transport proteins has been shown previously to influence aging via metabolic potential and a mechanism resembling caloric restriction. We report here a functional coupling of NHX-2 activity with nutrient uptake that results in long lived worms. Gene inactivation of nhx-2 by RNAi led to a loss of fat stores in the intestine and a 40% increase in longevity. The NHX-2 protein was coincidentally expressed with OPT-2, an oligopeptide transporter that is driven by a transmembrane proton gradient and that is also known to be involved in fat accumulation. Gene inactivation of opt-2 led to a phenotype resembling that of nhx-2, although not as severe. In order to explore this potential functional interaction, we combined RNA interference with a genetically encoded, fluorescence-based reagent to measure intestinal intracellular pH (pHi) in live worms under physiological conditions. Our results suggest first that OPT-2 is the main dipeptide uptake pathway in the nematode intestine, and second that dipeptide uptake results in intestinal cell acidification, and finally that recovery following dipeptide-induced acidification is normally a function of NHX-2. The loss of NHX-2 protein results in decreased steady-state intestinal cell pHi, and we hypothesize that this change perturbs proton-coupled nutrient uptake processes such as performed by OPT-2. Our data demonstrate a functional role for a Na+/H+ exchanger in nutrient absorption in vivo and lays the groundwork for examining integrated acid-base physiology in a non-mammalian model organism.

ATP-Binding Cassette Transporters↗
PubMed2003

Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues.

Gel-forming mucins are major contributors to the viscoelastic properties of mucus secretion. Currently, four gel-forming mucin genes have been identified: MUC2, MUC5AC, MUC5B, and MUC6. All these genes have five major cysteine-rich domains (four von Willebrand factor [vWF] C or D domains and one Cystine-knot [CT] domain) as their distinctive features, in contrast to other non-gel-forming type of mucins. The CT domain is believed to be involved in the initial mucin dimer formation and have very succinct relationship between different gel-forming mucins across different species. Because of gene duplication and evolutional modification, it is very likely that other gel-forming mucin genes exist. To search for new gel-forming mucin candidate genes, a "Hidden Markov Model"(HMM) was built from the common features of the CT domains of those gel-forming mucins. By using this model to screen all protein databases as well as the six-frame translated expression sequence tag and translated human genomic databases, we identified a locus located at the peri-centromere region of human chromosome 12 and the corresponding homologous region of mouse chromosome 15. We cloned the 3' end of this gene and its mouse homolog. We found one vWF C domain, one CT domain, and various mucin-like threonine/serine-rich repeats. Phylogenetic analysis indicated the close relationship between this gene and the submaxillary mucin from porcine and bovine. A polydispersed signal was observed on the Northern blot, which indicates very large mRNA size. Further analysis of the upstream genomic sequences generated from human and mouse genome projects revealed three additional vWF D domains and many mucin-like threonine/serine-rich repeats. The expression of this gene is restricted to the mucous cells of various glandular tissues, including sublingual gland, submandibular gland, and submucosal gland of the trachea. Based on the chronological convention, we have given the name MUC19 to the human ortholog and Muc19 to the mouse.

Amino Acid Sequence↗
PubMed2003

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Amino Acid Sequence↗
PubMed2003

Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon.

Colonic K+ secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca2+. However, the precise mechanisms by which this rise in Ca2+ leads to K+ efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] Ca2+-activated K+ channels. In dietary K+-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K+ channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca2+-activated K+ channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K+ secretion was studied on epithelial strips of rat proximal colon using unidirectional 86Rb+ fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net 86Rb+ secretion. The mucosal addition of CLT completely inhibited carbachol-induced net 86Rb+ secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K+, carbachol-induced 86Rb+ secretion was abolished in dietary K+-depleted animals. These results suggest that the rSK4 channel mediates K+ secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent K+ loss during dietary K+ depletion.

Amino Acid Sequence↗
PubMed2002

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels.

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.

Animals↗
PubMed2002

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene.

Salivary gland acinar cells shrink when Cl(-) currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl(-) channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl(-) channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl(-) currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca(2+)-calmodulin-dependent protein kinase II; however, the magnitude of the Ca(2+)-dependent Cl(-) current was unchanged in the Clcn3(-/-) animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3(-/-) mice. Our results demonstrate that the ClC-3 Cl(-) channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

Animals↗
PubMed2002

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells.

We used molecular biological and patch-clamp techniques to identify the Ca(2+)-activated K(+) channel genes in mouse parotid acinar cells. Two types of K(+) channels were activated by intracellular Ca(2+) with single-channel conductance values of 22 and 140 pS (in 135 mM external K(+)), consistent with the intermediate and maxi-K classes of Ca(2+)-activated K(+) channels, typified by the mIK1 (Kcnn4) and mSlo (Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo beta-subunits (Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each beta-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from beta(1) knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the beta(4)-subunit.

Alternative Splicing↗