Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis.

AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca(2+) concentration or pH. METHODS: To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca(2+) were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. RESULTS: SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca(2+) uptake into mitochondria, and K(ATP) channel regulators had no significant effect on intravesicular free Ca(2+) concentrations or vesicular pH. CONCLUSIONS/INTERPRETATION: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.

Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1.

The transmission of pain signalling involves the cytoskeleton, but mechanistically this is poorly understood. We recently demonstrated that the capsaicin receptor TRPV1, a non-selective cation channel expressed by nociceptors that is capable of detecting multiple pain-producing stimuli, directly interacts with the tubulin cytoskeleton. We hypothesized that the tubulin cytoskeleton is a downstream effector of TRPV1 activation. Here we show that activation of TRPV1 results in the rapid disassembly of microtubules, but not of the actin or neurofilament cytoskeletons. TRPV1 activation mainly affects dynamic microtubules that contain tyrosinated tubulins, whereas stable microtubules are apparently unaffected. The C-terminal fragment of TRPV1 exerts a stabilizing effect on microtubules when over-expressed in F11 cells. These findings suggest that TRPV1 activation may contribute to cytoskeleton remodelling and so influence nociception.

Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells.

To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking signal variants in mammalian cells, flow cytometry to sort cells based on trafficking phenotype, and quantitative trafficking assays to measure the efficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate that small changes in the local sequence context dramatically alter signal strength, generating a broad spectrum of trafficking phenotypes. Finally, using sequences from our screen, we found that the potency of di-lysine, but not di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha-COP.

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter.

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.

A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels.

Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.

Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p.

GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.

A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption.

We have cloned four novel members of the CLC family of chloride channels from Arabidopsis thaliana. The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C. (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7 putative chloride channels from mammalia. AtCLC transcripts are broadly expressed in the plant. Similarly, antibodies against the AtCLC-d protein detected the protein in all tissues, but predominantly in the silique. AtCLC-a and AtCLC-b are highly homologous to each other ( approximately 87% identity), while being approximately 50% identical to either AtCLC-c or AtCLC-d. None of the four cDNAs elicited chloride currents when expressed in Xenopus oocytes, either singly or in combination. Among these genes, only AtCLC-d could functionally substitute for the single yeast CLC protein, restoring iron-limited growth of a strain disrupted for this gene. Introduction of disease causing mutations, identified in human CLC genes, abolished this capacity. Consistent with a similar function of both proteins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein. This suggests that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.

A common molecular basis for three inherited kidney stone diseases.

Kidney stones (nephrolithiasis), which affect 12% of males and 5% of females in the western world, are familial in 45% of patients and are most commonly associated with hypercalciuria. Three disorders of hypercalciuric nephrolithiasis (Dent's disease, X-linked recessive nephrolithiasis (XRN), and X-linked recessive hypophosphataemic rickets (XLRH)) have been mapped to Xp11.22 (refs 5-7). A microdeletion in one Dent's disease kindred allowed the identification of a candidate gene, CLCN5 (refs 8,9) which encodes a putative renal chloride channel. Here we report the investigation of 11 kindreds with these renal tubular disorders for CLCN5 abnormalities; this identified three nonsense, four missense and two donor splice site mutations, together with one intragenic deletion and one microdeletion encompassing the entire gene. Heterologous expression of wild-type CLCN5 in Xenopus oocytes yielded outwardly rectifying chloride currents, which were either abolished or markedly reduced by the mutations. The common aetiology for Dent's disease, XRN and XLRH indicates that CLCN5 may be involved in other renal tubular disorders associated with kidney stones.

Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease.

We have cloned a novel member of the CLC chloride channel family from rat brain, rCLC-5. The cDNA predicts a 83-kDa protein belonging to the branch including CLC-3 and CLC-4, with which it shares approximately 80% identity. Expression of rCLC-5 in Xenopus oocytes elicits novel anion currents. They are strongly outwardly rectifying and have a conductivity sequence of NO3- > Cl- > Br- > I- >> glutamate-. Although CLC-5 has consensus sites for phosphorylation by protein kinase A, raising the intracellular cAMP concentration had no effect on these currents. Currents were also unchanged when rCLC-5 was coexpressed with rCLC-3 and rCLC-4, either singly or in combination. rCLC-5 is expressed predominantly in kidney and also in brain, lung, and liver. Along the nephron, rCLC-5 message is detectable in all tubule segments investigated, but expression in the glomerulus and the S2 segment of the proximal tubule is low.

Properties of voltage-gated chloride channels of the ClC gene family.

We review the properties of ClC chloride channels, members of an expanding gene family originally discovered by the cloning of the ClC-0 chloride channel from Torpedo electric organ. There are at least nine different ClC genes in mammals, several of which seem to be expressed ubiquitously, while others are expressed in a highly specific manner (e.g. the muscle-specific ClC-1 channel and the kidney-specific ClC-K channels). The newly cloned rat ClC-4 is strongly expressed in liver and brain, but also in heart, muscle, kidney and spleen. ClC chloride channels are structurally unrelated to other channel proteins and have twelve putative transmembrane domains. They function as multimers with probably four subunits. Functional characterization is most advanced with ClC-0, ClC-1 (mutations which cause myotonia) and ClC-2, a swelling-activated chloride channel. Many of the new ClC family members cannot yet be expressed functionally.

Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements.

A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P(i) + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes.