B.Sc., University of Toronto, 1978
Ph.D., University of Toronto, 1984
University of Calgary 1984-87
University of Liverpool, UK visiting fellowship,
McMaster Building, HSC, Room 3203B
Epithelial Chloride Channels
and Their Role in Cell Physiology and Pathophysiology
|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.
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):
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,
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,
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):
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: