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Terry Machen, Grischa Chandy,
Minnie Wu, Michael Grabe, and Hsiao-Ping Moore. (2001). CFTR and H+
permeability in regulation of Golgi pH. Proceedings of the
International Symposium on HC03- and Cystic Fibrosis, P.Quinton, ed.
J. Pancreas. 2(4), 229-236.
This paper reviews experiments from this
lab that have tested the hypothesis that pH of the Golgi (pH(G)) of cystic
fibrosis (CF) airway epithelial cells is alkaline compared to normal, that this
altered pH affects sialyltransferase and other Golgi enzymes controlling
biochemical composition of the plasma membrane and that altered surface
biochemistry increases bacterial binding. We generated a plasmid encoding a
modified green fluorescence protein-sialyltransferase (GFP-ST) chimera protein
that was pH-sensitive and localized to the Golgi when transfected into HeLa
cells and also CF and normal or cystic fibrosis transmembrane conductance
regulator- (CFTR)-corrected airway epithelial cells. Digital imaging microscopy
of these Golgi-localized probes showed that there was no correlation between
pH(G) (6.4-7.0) and the presence of CFTR, whether cells were in
HCO(3)(-)/CO(2)-containing or in HCO(3)(-)/CO(2)-free solutions. Activation of
CFTR by raising cell [cAMP] had no effect on pH(G). Thus, CFTR seemed not to be
involved in controlling pH(G). Experiments on HeLa cells using an
avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent
biotin indicated that even in cells that do not express CFTR, Cl(-) and K(+)
conductances of the Golgi and other organelle membranes were large and that pH(G)
was controlled solely by the H(+) v-ATPase countered by a H(+) leak. A
mathematical model was applied to these and other published data to calculate
passive H(+) permeability (P(H+)) of the Golgi, endoplasmic reticulum,
trans-Golgi network, recycling endosomes and secrety granules from a variety of
cells. An organelle's acidity was inversely correlated to its calculated P(H+).
We conclude that the CFTR plays a minor role in organelle pH regulation because
other (Cl(-) and K(+)) channels are present in sufficient numbers to shunt
voltages generated during H(+) pumping. Acidity of the Golgi (and perhaps other
organelles) appears to be determined by the activity of H(+) pumps countered by
H(+) leaks. |
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