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Biotechnology↗
Biomedical subjects
C R Redhead
Publications and source records attributed to C R Redhead.
A ubiquitous 64-kDa protein is a component of a chloride channel of plasma and intracellular membranes.
Chloride channels are present in the plasma and intracellular membranes of most cells. Previously, using the ligand indanyloxyacetic acid (IAA), we purified four major proteins from bovine kidney cortex membrane vesicles. These proteins gave rise to chloride channel activity when reconstituted into phospholipid vesicles. Two of these proteins (97 and 27 kDa) were found to be drug-binding proteins by N-terminal sequence analysis. Antibodies raised to the 64-kDa protein stained only this protein on immunoblots, and only this protein was present after purification on an immunoaffinity column. In addition, these same antibodies were able to deplete IAA-94 inhibitable chloride channel activity from solubilized kidney membranes. Of fractions obtained from the gel filtration of solubilized kidney membranes, only those containing this 64-kDa protein exhibited measurable chloride channel activity. Immunoblots of a variety of species and cell types, both epithelial and nonepithelial, revealed that this protein is ubiquitous and highly conserved. Immunocytochemistry in CFPAC-1 cells revealed staining for this protein on the apical plasma membrane and in the membranes of intracellular organelles. These results demonstrate that the integral membrane protein p64 is a component of chloride channels present in both epithelial plasma membrane and the membranes of intracellular organelles.
Amino Acid Sequence↗
Control of intracellular pH in rat calvarial osteoblasts: coexistence of both chloride-bicarbonate and sodium-hydrogen exchange.
Intracellular pH was monitored continuously in cultured rat calvarial osteoblasts using the pH-sensitive fluorescent dye bis carboxyethyl carboxyfluorescein (BCECF), loaded into the cells as its membrane permeant ester. Recovery from an intracellular acid load generated by exposure to NH4Cl was unaffected by the anion exchange inhibitors 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disuphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene 2,2'-disuphonic acid (DIDS) (100 microM), but blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM) and dependent on external sodium, suggesting that recovery is brought about by a sodium-hydrogen exchanger in the plasma membrane. The cells do, however, possess a SITS-sensitive chloride-bicarbonate exchanger, because iso-osmotic replacement of chloride by gluconate leads to intracellular alkalinization, that is inhibited by SITS, but independent of external sodium. Parathyroid hormone brings about an intracellular acidification, which may be due to an inhibiton of sodium-hydrogen exchange.
Acid-Base Equilibrium↗
Ionic regulation of intracellular pH in rat calvarial osteoblasts.
1. Intracellular pH of cultured rat calvarial osteoblasts was monitored continuously using the pH-sensitive fluorescent probe bis-carboxyethyl carboxyfluorescein. 2. Recovery from an intracellular acid load brought about by exposure to ammonium chloride was dependent on external sodium and blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM), indicating the presence of a plasma membrane sodium-hydrogen exchanger. 3. A SITS- (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid) sensitive alkalinization occurred on the isosmotic replacement of external chloride by gluconate, suggesting the presence of chloride-bicarbonate exchange. 4. The dependence of the rate of sodium-hydrogen exchange on external sodium followed first-order kinetics, but the rate of exchange appeared to be sensitive to intracellular pH. 5. The rate of alkalinization brought about by the isosmotic replacement of chloride was sensitive to external bicarbonate concentration, but independent of external sodium. 6. Sodium-hydrogen exchange appeared to be inhibited and chloride-bicarbonate exchange stimulated by 1-34 parathyroid hormone.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗