Deborah Joyce Nelson, Ph.D.

Research Summary

Function of Ion Channels in Excitation-Secretion Coupling

Research in the laboratory over the past ten years has further explored ion channel-mediated signal transduction in non-excitable cells focusing on regulation via intracellular protein-protein interactions. Using recent examples of studies conducted in the laboratory, these interactions can subserve vastly different cellular functions which may gate or open a channel as in the case of the G protein coupled K channel (GIRK or Kir 3.X) (1) or the CaMKII-activated chloride channel (2,3). Protein-protein interactions may modulate the time a channel spends in the open state as with the interaction of members of the SNARE protein family with the CFTR (Cystic Fibrosis Transport Regulator) chloride channel (4-6). Conversely, a complex of regulatory proteins may play a concerted role in inhibiting channel open time as is the case with annexin IV and CaMKII in the regulation of the CaMKII –activated chloride channel (7,8). And finally, ion channels may be held together in regulatory networks or membrane rafts via interactions with the actin cytoskeleton (9). We have used as our target proteins both K and Cl channels and have studied protein mediated channel regulation in both classes of proteins recognized as mediators of membrane potential stabilization.

CFTR Chloride Channel Modulation by Vesicle Trafficking Proteins

Accumulating evidence suggests that many ion channels reside within a multiprotein complex that contains kinases and other signaling molecules. CFTRis an example of such a channel. CFTR is activated by cAMP dependent kinase when two nucleotide binding domains are bound with ATP. Over the past few years, my laboratory has collaborated with the laboratory of Dr. Kevin Kirk at the University of Alabama at Birmingham to explore protein-protein interactions between CFTR and vesicle trafficking proteins of the class used to control neurotransmitter release in neuroendocrine cells. We have established that three of these proteins, namely syntaxin, munc-18 and SNAP 23 all interact to modulate channel open time. The interaction of syntaxin is highly specific, recognizing a segment of some 20 amino acids in the N-terminal domain of CFTR to inhibit channel opening. The binding of munc-18 and SNAP 23 regulate the affinity of the binding interaction between syntaxin and CFTR and, thereby, channel open time. We have established that the interaction is stoichiometric and involves direct protein-protein interactions rather than changes in protein trafficking. This paradigm of membrane trafficking proteins, syntaxin 1A, SNAP 23 and munc 18, regulating the activity of the proteins which are at the cell surface has been subsequently shown for a number of channel and transport proteins and our studies provided the first evidence that such a interaction exists and that the interaction is direct.

Chloride Channel Biology

The activation of chloride channels subserves a multiplicity of cellular functions including membrane potential stabilization, volume regulation, salt and water balance, and intracellular vesicle acidification. Recent work in the laboratory has focused on the cloning, expression, and regulation of one of the most important of the voltage dependent chloride channels, ClC-3. While broad expression and physiological importance of ClC-3 has been established, the mechanism of channel activation has remained elusive. In a recent study, my laboratory has characterized the activation pathway for ClC-3 when it is expressed in the plasma membrane and has shown its gating to be dependent upon phosphorylation by the multifunctional, calcium/calmodulin dependent kinase, CaMKII. In earlier studies on the endogenous channel expressed in cell lines derived from the gastrointestinal system we were able to show that the channel was regulated by inositol phosphates and the calcium/phospholipid dependent protein annexin IV (2,7). On-going studies are directed at the determination of channel oligomeric structure in the plasma membrane as well as cytoplasmic compartments and preliminary data suggests that the channel can function in two oligomeric states dependent upon the site of expression. If this turns out to be the case, then ClC-3 will be the only channel that is capable of functional expression in two different oligomeric forms. Recent studies in the laboratory also demonstrate that regulation of ClC-3 involves a cytoskeletal scaffolding that localizes the activating kinase in close proximity to its target channel domain.

Macrophage Function: Secretion and Particle Uptake

The final component of the research agenda within the laboratory focuses on the regulation of particle uptake and secretion in the macrophage bactericidal response. Our recent studies have determined that unlike the neuroendocrine cell, secretion in the macrophage is highly dependent upon activated G proteins (10). Calcium plays only a modulatory role, enhancing the gain on secretion presumably by mobilizing vesicles from a ready reserve pool. This model for secretion is vastly different from that present in cells of neuroendocrine origin where secretion is determined in toto by a calcium dependent mechanism. The ability to selectively mobilize membrane bound granule/vesicle proteins into the external environment is central to the role of the macrophage in the inflammatory response. Surface receptor ligation by invading microorganisms initiates the immune response via the formation of a plasma membrane bound phagosome. The content of the phagosome is determined primarily by the contents of the cytoplasmic granules that discharge into it immediately following particle ingestion. The cellular fate of the fully mature phagosome, a subset of the intracellular vesicle population present in the macrophage, had not been determined until the publication of our recent study demonstrating quantal release of free radicals which accompanies phagosomal recycling to plasma membrane sites (11).

Future Investigations and Directions

Studies on-going in the laboratory are directed at the subcellular localization of the regulatory proteins involved in the activation of the chloride channel ClC-3. In parallel, we are continuing our productive collaboration with Dr. Kevin Kirk at the University of Alabama at Birmingham further exploring protein-protein interactions in the regulation of CFTR. We have extended our studies to including a mutational analyis of the binding partners within the SNARE complex in epithelial cells and CFTR in an attempt to augment the channel trafficking defect that is present in the disease of cystic fibrosis. We are also involved in an active collaborative relationship with Dr. Clive Palfrey here at the University where we are exploring the involvement of the GTP-ase, dynamin in the regulation of both particle uptake and phagosomal recycling in the activated macrophage. Finally, we are exploring the mechanism of channel gating in the G protein activated K channel K. It is our hypothesis that the C terminal domains of the multisubunit structure interact to form a binding pocket stabilizing activation by the heterotrimeric G protein subunits Gbg.

Selected Papers

Li, C., Dandridge, K.S., Di, A., Marrs, K.L., Harris, E.L., Koushik, R., Jackson, J.S., Makarova, N.V., Fugiwara, Y., Farrar, P.L., Nelson, D.J., Tigyi, G.J., and Naren, A.P. 2005  Lysophosphatidic acid inhibition of diarrhea through CFTR-dependent protein interactions.  J. Exp. Med.  202:975-986.

Di, A., Brown, M.E., Deriy, L.V., Li, C., Szeto, F.L., Chen, Y., Huang, P., Tong, J., Naren, A.P.,  Bindokas, V., Palfrey, H.C., and Nelson, D.J. 2006.  CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.  Nature Cell Biology  8:933-944.

Faculty of 1000 Biology: evaluations for Di A et al Nat Cell Biol 2006 Sep 8 (9):933-44 http://www.f1000biology.com/article/id/1040460/evaluation

News and Views for this paper:
Swanson, J. 2006.  CFTR: helping to acidify macrophage lysosomes.  Nature Cell Biology 8:908-909.
Burroughs Wellcome Science 5/ Topical Updates 1081 Citation

Wang, X.Q., Deriy, L.V., Foss, S., Huang, P., Lamb, F.S., Kaetzel, M.A., Bindokas, V., Marks, J.D., Nelson, D.J. 2006.  ClC3 channels modulate excitatory synaptic transmission in hippocampal neurons. Neuron52: 321-333.  
               
Nature Research Highlights Citation for this paper: Housekeeper has two jobs.  Nature 444:4 (2006)
               
Ganeshan, R., Nowotarski, K., Di, A., Nelson, D.J., and Kirk, K.L. 2007. An N-WASP inhibitor decreases CFTR surface expression and CFTR-mediated chloride currents. Biochimica et Biophysica Acta – Molecular Cell Research1773(2):192-200.

Li, C.,  Krishnamurthy, P.C.,  Penmatsa, H., Marrs, K.L., Wang, X.Q., Zaccolo, M., Jalink, K., Li, M., Nelson, D.J., Schuetz, J.D., and  Naren, A.P.  2007.  Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.  Cell 131: 940-951.

Claud, E.C., Lu, J., Wang,,XQ., Abe, M., Petrof , E.O., Sun, J., Nelson, D.J., Marks, J.D., Jilling, T. 2008.  Platelet-activating Factor induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells.  Am J. Physiol. Gastrointest. Liver Physiol. 294:G1191-2000.

Mitchell, J., Wang, X., Zhang, G., Gentzsch, M., Nelson, D.J., and Shears, S.B.  2008.  An expanded biological repertoire for Ins(3,4,5,6)P4 through its modulation of ClC-3 function.    Current Biology 18(20):1600-5.

Deriy, L.V., Gomez, E.A.,  Jacobson, D.A., Wang, X..,  Hopson, J.A., Liu, X.Y., Zhang, G., Bindokas, V.P.,  Philipson, L.H., and  Nelson,, D.J.  2009.  The granular chloride channel ClC-3 is permissive for insulin secretion. Cell Metabolism  10:316-323.

Deriy, L.V., Gomez, E.A., Zhang, G., Beacham, D., Hopson, J.A., Gallan, A.J., Shevchenko, P., Bindokas, V.P., and Nelson, D.J. 2009.  Disease causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages.  J. Biol. Chem. 284:35926-38.

Mitterreiter, S., Page, R.M.,  Kamp, F., Hopson, J.A., Winkler, E., Ha, H.-R., Hamid, R., Herms, J., Mayer, T.U., Nelson, D.J., Steiner, H., Stahl, T., Zeitschel, U., Rossner, S., Haass, C., and  Lichtenthaler, S.F. 2010.  Bepridil and amiodarone simultaneously target the Alzheimer’s disease b- and g-secretase via distinct mechanisms.  J. Neuroscience 30:8974-83.

Penmasta, H., Zhang, W., Yarlagadda, S., Li, C., Conoley, V.G., Yue, J., Bahouth, S.W., Buddington, R.K., Zhang, G., Nelson, D.J., Sonecha, M.D.,  Manganiello, V., Wine, J.J., Naren, A.P. 2010.  Compartmentalized cAMP at the plasma membrane clusters PDE3A and CFTR into microdomains.   Molecular Biology of the Cell 21:1097-110.