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Systemic mutation of histidine residues at the SDHC and SDHD subunits of Succinate:ubiquinone oxidoreductase or the cytochrome b556 complex II was undertaken to determine the residues that function as axial ligands for the heme moiety. Results reveal that the likely axial ligands are the histidine residues at position 84 of SDHC and position 71 of SDHD. Mutation of the histidine residue at position 30 of SDHD prevented the assembly of the complex.

Genetic research shows that the Na-K-Cl cotransporter molecule of bovine aortic endothelial cells consists of a 1,201-amino acid protein chain with 12 putative transmembrane segments that maps closely with the NKCC1 amino acid sequence. cDNA library data was screened to ascertain sequencing information. Sequence alignment with deduced primary structures for the cotransporter molecule are given.

Previous studies have reported that a tryptophan substitution on the alphaC418 position at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor (AChR) resulted in a 23-fold decrease in the closing rate constant of the (AChR). Herein, measurements of the open and closed rate constant in three multiple mutants for tryptophan revealed that tryptophan substitutions produced significant effects on the channel gating kinetics. Data indicated that the number of substitutions was directly related to the open probability of the channel. Kinetic equations that describe the channel gating mechanisms are presented.

Putney, Luanna K., Cecile Rose T. Vibat, and Martha E. O'Donnell. Intracellular C1 regulates Na-K-C1 cotransport activity in human trabecular meshwork cells. Am. J. Physiol. 277 (Cell Physiol. 46): C373-C383, 1999.--The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([[Cl]i.sub.]) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [[Cl].sub.i]. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [[Cl].sub.i]. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [[Cl].sub.i] results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [[Cl].sub.i], as well as elevating [[Cl].sub.i], on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [[Cl].sub.i] is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [[Cl].sub.i]. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [[Cl].sub.i] may participate in modulating outflow facility across the TM. glaucoma; aqueous outflow; intracellular volume; chloride channel; nifiumic acid