The most prevalent form of diabetes (type II; 90% of cases) is characterized by insulin resistance in peripheral tissues and/or a deficiency of insulin due to the failure of pancreatic beta-cells to secrete insulin. Considerable progress has been made in determining many features of secretagogue-regulated insulin secretion, particularly the role of plasma membrane ion channels in the process ¹.  However, we are still at an early stage in defining the molecular mechanisms involved in exocytosis of the insulin granule, and the possible role of granule membrane ion channels in this process. Previously, we showed that the chloride channel ClC-3 is functionally expressed in the membrane of insulin-containing granules. Functional studies in isolated beta-cells showed that activation of ClC-3 is permissive for insulin secretion. This is due, at least in part, to the promotion of granular acidification; various strategies to abolish acidification disrupt secretion in a similar manner. These observations are part of a burgeoning literature on the important role played by vesicular ion channels in secretion, as well as the more specific requirement for vesicle acidification in several cases. Recently, we have extended our findings to the ClC-3 knockout mouse.  Preliminary data presented below indicate that beta-cells from this model are defective in exocytosis and animals exhibit aberrant glucose tolerance, indicating that they may become diabetic.  Current studies in the laboratory build on this foundation and are targeted at unraveling in molecular detail the role of ClC-3 chloride channels in beta-cell secretion. 

     Until recently anion channels have received relatively little attention compared to their cation channel cousins in the regulation of beta cell secretion. The complexity of chloride channels became apparent with the cloning of an evolutionarily-conserved family of Cl^- channels (ClCs), as well as the discovery of other channel families that principally carry Cl^- (e.g. CFTR, Ca²⁺-activated Cl^- channels) ². ClC-3 is a prominent member of the former category and is widely distributed in mammalian tissues. It can assume either a plasma membrane or intracellular membrane localization, and has been proposed as the channel mediating acidification in endosomes ³. Mice lacking ClC-3 exhibit a variety of defects, most prominently postnatal degeneration of the hippocampus and consequent behavioral deficits. ClC-3 is present on synaptic vesicles and may play a role in glutamatergic neurotransmission release; indeed, we recently showed that ClC-3 likely shapes synaptic currents in developing hippocampal neurons in part by studying clc-3^-/-4. ClC-3 channels have extended N- and C-termini. The N-terminus may contain important phosphorylation sites while the C-terminus contains “CBS” domains that likely bind adenine nucleotides ^{5, 6}. We have spent several years investigating and continue to study this channel at the molecular, cellular and organismal level. mice

    Mutations in ClC channels have been linked to a diversity of disease states in tissues ranging from kidney to muscle ^{2, 7-11}.  Jentsch, in a recent review of pathologies linked to the members of the two ClC subfamilies expressed in the intracellular compartment, noted that genetically engineered mice lacking specific ClC proteins aided in the identification of the physiological functions of vesicular ClCs ¹².  The two subfamilies of ClCs located in synaptic vesicles, endosomes, and lysosomes (including ClC-3 through 7) regulate vesicular acidification.  It should be noted that ClC-3, unlike other members of the two subfamilies, is expressed at both intracellular and plasma membrane sites which may be a result of differential isoform expression (there are at least 3 distinct isoforms of ClC-3 that have been identified ^{13, 14}.  Extensive, specific neurodegeneration has been documented in the three ClC-3 knockout mice that have been made ^15-17. Loss or disruption of ClC-5 results in Dent's disease characterized by proteinuria and kidney stones ^{7, 18}, symptoms that are similar in both mice and humans.   ClC-6 appears to be a candidate gene for mild forms of neuronal ceroid lipofusinosis ¹⁹, while loss of ClC-7 In humans and mice gives rise to osteopetrosis like symptomatology, blindness as well as lysosomal storage disease ^{20, 21}.  To date no human disease causing mutations have been identified in either the ClC-3 or -4 gene.  Our previous collaborative studies on the functional analysis of ClC-3 in pancreatic beta-cells ²² along with the preliminary experiments on pancreatic beta cells isolated from ClC-3 null animals provide the rationale for further investigations into the role of  in the regulation of insulin release in human disease.

 

 REFERENCES

 

1.         Rorsman, P. & Renstrom, E. Insulin granule dynamics in pancreatic beta cells. Diabetologia 46, 1029-1045 (2003).

2.         Jentsch, T.J., Stein, V., Weinreich, F. & Zdebik, A.A. Molecular structure and physiological function of chloride channels. Physiol Rev 82, 503-568 (2002).

3.         Hara-Chikuma, M. et al. ClC-3 Chloride Channels Facilitate Endosomal Acidification and Chloride Accumulation. The Journal of biological chemistry 280, 1241-1247 (2005).

4.         Wang, X.Q. et al. CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons. Neuron 52, 321-333 (2006).

5.         Wellhauser, L. et al. Nucleotides bind to the C-terminus of ClC-5. Biochem J 398, 289-294 (2006).

6.         Meyer, S., Savaresi, S., Forster, I.C. & Dutzler, R. Nucleotide recognition by the cytoplasmic domain of the human chloride transporter ClC-5. Nature structural & molecular biology 14, 60-67 (2007).

7.         Gunther, W., Piwon, N. & Jentsch, T.J. The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease. Pflugers Arch 445, 456-462 (2003).

8.         Hubner, C.A. & Jentsch, T.J. Ion channel diseases. Hum Mol Genet 11, 2435-2445 (2002).

9.         Estevez, R. & Jentsch, T.J. CLC chloride channels: correlating structure with function. Curr Opin Struct Biol 12, 531-539 (2002).

10.       Boettger, T. et al. Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4. Nature 416, 874-878 (2002).

11.       Jentsch, T.J. Chloride channels are different. Nature 415, 276-277 (2002).

12.       Jentsch, T.J. Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters. The Journal of physiology 578, 633-640 (2007).

13.       Gentzsch, M. et al. The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with CFTR-interacting PDZ proteins. Journal of Biological Chemistry 278, 6440-6449 (2002).

14.       Ogura, T. et al. ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC. FASEB Journal 16, 863-865 (2002).

15.       Stobrawa, S.M. et al. Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29, 185-196 (2001).

16.       Dickerson, L.W. et al. Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels. Brain Res 958, 227-250 (2002).

17.       Yoshikawa, M. et al. CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis. Genes Cells 7, 597-605 (2002).

18.       Gunther, W., Luchow, A., Cluzeaud, F., Vandewalle, A. & Jentsch, T.J. ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytically active kidney cells. Proceedings of the National Academy of Sciences USA 95, 8075-8080 (1998).

19.       Poet, M. et al. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proceedings of the National Academy of Sciences of the United States of America 103, 13854-13859 (2006).

20.       Kornak, U. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205-215 (2001).

21.       Lange, P.F., Wartosch, L., Jentsch, T.J. & Fuhrmann, J.C. ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. Nature 440, 220-223 (2006).

22.       Barg, S. et al. Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. Journal of cell science 114, 2145-2154 (2001).