Christine E. Bear Professor

B.Sc., University of Toronto, 1978
Ph.D., University of Toronto, 1984
University of Calgary 1984-87
University of Liverpool, UK visiting fellowship, 1986
Elizabeth McMaster Building, HSC, Room 3203B
416-813-5981
bear@sickkids.ca

Epithelial Chloride Channels and Their Role in Cell Physiology and Pathophysiology



Research Synopsis
 
Chloride channels contribute to critical biological functions such as the regulation of electrical excitability in muscle and neurons, fluid transport in epithelial tissues and pH regulation in intracellular organelles. Mutations in genes which encode chloride channels lead to human diseases such as Congenital Myotonia, Cystic Fibrosis, Dent’s disease of the kidney and Congenital Osteopretosis. We are primarily interested in defining the molecular basis of the diseases that arise from mutations in specific chloride channel genes.

At present, our specific studies focus on understanding the molecular and structural basis for Cystic Fibrosis, the disease caused by mutations in the gene, CFTR. Our group developed the means for purification and functional reconstitution of the protein product of CFTR (Bear, C. et al. Cell 1992). Further, we showed in electrophysiological studies in planar lipid bilayers, that wild type CFTR functions as a chloride channel and as an ATPase and certain disease causing mutations in CFTR cause specific defects in these functions (Li, C. et al. Nature Genetics 1993) (Li, C. et al. J. Biol. Chem. 1996) (Pasyk, E. et al. J. Biol. Chem. 1998), (Kogan, I. et al. J. Biol. Chem. 2001). Recently, we have developed novel methods for assessment of the quaternary structure of CFTR in cell membranes obtained from native tissues and the separation and reconstitution of each structure (Ramjeesingh, M. et al., Biochem. J. 1999) (Ramjeesingh, M. et. al. Biochemistry, 2000) . We found that while monomeric CFTR is the minimal functional unit, the protein self-associates to form dimers at the plasma membrane. We are currently using our reconstitution system to determine the functional consequences of this self association and the impact of disease-causing mutations on this process.

According to studies by our group and other research groups, it is clear that the severity of Cystic Fibrosis relates not only to the molecular consequences of disease causing mutations but also to the function of "modifying" genes which may complement the chloride channel function CFTR (Rozmahel R. et al. Nature Genetics 1993) (Kent, G. et al. J. Clin. Invest. 1999). Recently, we have determined using immunofluorescence with confocal and electron microscopy that members of a large family of chloride channels, the ClC family of chloride channels are localized appropriately in the plasma membrane of epithelial cells and function as chloride channels at this site (Mohammad-Panah, R. et al. J. Biol. Chem., 2001) . At present, we are assessing the capacity of these other channels to functionally complement CFTR in CF-affected tissues. Furthermore, we are studying the biochemical and biophysical properties of these channels using the tools described in the preceding paragraphs. In fact, we have purified and functionally reconstituted monomeric, dimeric and tetrameric ClC-2 and determined that unlike CFTR, a dimer is the minimum functional unit of this channel forming protein (Ramjeesingh, M. et al. Biochemistry, 2000). Our long term goal is to obtain a high resolution structure of these chloride channels in order to confirm and define basic features, such as the quaternary structure, the channel pore and the basis for the interaction between the pore and regulatory domains.
 


Selected Publications

Grimard V., Li C., Ramjeesingh M., Bear C.E., Goormaghtigh E., Ruysschaert J-M. Phosphorylation induced conformational changes of CFTR monitored by ATR-FTIR and fluorescence spectroscopy. J. Biol. Chem. 279(7):
5528-36, 2004.

Ramjeesingh M., Ugwu F., Li C., Dhani S., Huan LJ., Wang Y. and Bear C.E. Stable dimeric assembly of the second membrane spanning domain of CFTR (cystic fibrosis transmembrane conductance regulator) reconstitutes
a chloride selective pore.
Biochem J. 375 (3): 633-41, 2003.

Ramjeesingh M., Kidd J.F., Huan L.J., Wang Y. and Bear C.E. Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma
membrane.
Biochem. J. 374 (3): 793-7, 2003.

Mohammad-Panah R., Harrington R., Dhani S., Ackerley C.A., Huan L.-J.,
WangY. and Bear C.E.  The chloride channel ClC-4 contributes to
endosomal acidification and trafficking.
J. Biol. Chem. 278 (31): 29267-77, 2003.

Kogan I., Ramjeesingh M., Kidd J.F., Wang Y.,  Bear C.E. CFTR Directly
mediates nucleotide-regulated  glutathione flux.
EMBO J.  22 (9): 1981-9, 2003.

Dhani SU, Mohammada-Panah R., Ahmed N., Ackerley C., Ramjeesingh M.
and Bear C.E.   Evidence for a functional interaction between the ClC-2
chloride channel and the retrograde motor dynein complex.
 J. Biol. Chem.
278 (18): 16262-70,  2003.

Mohammad-Panah R, Ackerley C, Rommens J, Choudrury M, Wang Y, Bear CE. The chloride channel ClC-4 co-localizes with CFTR and may mediate chloride flux across the apical membrane of intestinal epithelia. J. Biol. Chem. Oct 23 [epub ahead of print], 2001.

Ramjeesingh M, Li C, Kogan I, Wang Y, Huan L-J, Bear CE. A monomer is the minimum functional unit for channel and ATPase activity of the Cystic Fibrosis Transmembrane Conductance Regulator. Biochemistry 40(35): 10700-6, 2001.

Kogan I, Ramjeesingh M, Huan L-J, Wang Y, Bear CE. Perturbation of the pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) inhibits its ATPase activity. J. Biol. Chem. 276(15): 11575-81, 2001.

Mohammad-Panah R, Gyömörey K, Rommens J, Choudhury M, Li C, Wang Y, Bear CE. ClC-2 contributes to native chloride secretion by a human intestinal cell line, Caco-2. J. Biol. Chem. 276(11): 8306-13, 2001.

Ramjeesingh M, Li C, Huan L-J, Garami E, Wang Y, Bear CE. Quaternary structure of the chloride channel ClC-2. Biochemistry 39 (45): 13838-47, 2000.

Ramjeesingh M, Li C, Garami E, Huan L-J, Galley K, Wang Y, Bear CE. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR. Biochemistry 38 (5): 1463-8, 1999.

Pasyk E, Morin X, Zeman P, Garami E, Galley K, Wang Y, Bear CE, Role of a Conserved Hydrophobic Region of the R Domain of CFTR in Processing and Function. J. Biol. Chem. 273: 31759-31764, 1998.

Ramjeesingh M, Huan L-J, Wilschanski M, Durie P, Li C, Gyomorey K, Wang Y, Kent G, Tanswell KA, Cutz E, Ackerley C, Bear CE. Assessment of the Efficacy of In Vivo CFTR Protein Replacement Therapy in CF Mice. Human Gene Therapy 9: 521-528, 1998.

Kent G, Grisenbach U, Huan L-J, Bear CE, McKerlie CA, Iles R, O’Brodovich H, Ackerley CA, Tsui LC, Buchwald M, Tanswell K. Lung Disease in Mice with Cystic Fibrosis. J. Clin. Invest. 100: 3060-3069, 1997.

Li C, Ramjeesingh M, Wang W, Lee D, Garami E, Hewryk M, Galley K, Bear CE. ATPase Activity of Purified, Reconstituted CFTR. J. Biol. Chem. 271: 28463-28468, 1996.

   

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