Negatively-charged Amino Acids Research Articles

Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.

Site-directed mutagenesis of the sodium-potassium-ATPase: Consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains

Site-directed mutagenesis was used to examine the importance of five carboxyl-containing amino acids localized in the putative membrane-spanning regions of the Na,K-ATPase (i.e., E327, E778, D803, D807, and D925 of the rat alpha 2 isoform). The substitutions were introduced into a cDNA encoding a ouabain-resistant isoform (i.e., rat alpha 2* which was mutated to encode a ouabain-resistant isoform), and the effect of these substitutions on Na,K-ATPase function was assessed by screening the altered enzymes for their ability to confer ouabain resistance when expressed in otherwise ouabain-sensitive cells. The expression of the alpha isoform containing certain substitutions at positions 327 and 925 was able to confer ouabain resistance to HeLa cells while the expression of rat alpha 2* containing substitutions at positions 778, 803, and 807 was not. In particular, amino acids in each of these positions were substituted with leucine to evaluate the importance of the carboxyl-containing side chain. The ability of rat alpha 2* containing E327L and D925L to confer ouabain resistance to HeLa cells indicates that neither the negative charge nor the oxygen-containing side chain is absolutely essential for overall function in this position. In contrast, the inability of rat alpha 2* carrying E778L, D803L, and D807L to confer ouabain resistance suggests that the naturally occurring amino acid may be more critical structurally and/or functionally for the Na,K-ATPase. Other more conservative substitutions introduced to further characterize the role of particular amino acid side chains include E327D, E327Q, D803N, D803E, and D925N.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping functional regions of the segment-specific transcription factor Krox-20

Krox-20, a zinc finger transcription factor with similarity to Sp1, is likely to play an important role in the development of the vertebrate central nervous system. A knowledge of its molecular properties will help to understand its physiological functions. We have therefore performed a structure-function analysis of the protein to identify the regions involved in DNA-binding and transcriptional activation. Our data suggest that only the zinc fingers are required for high affinity, specific DNA-binding. Transcriptional activation was not affected by deletion of the C-terminal tail of the protein. In contrast, deletion of the N-terminal half, upstream of the zinc fingers, completely abolished transactivation without affecting DNA-binding or nuclear localization. Two transcriptional activation domains were identified in this region. They cooperate to establish full activity. They are rich in negatively-charged amino acids and are therefore may constitute acidic activation domains. Comparative analysis of the amino acid sequences of several zinc finger proteins belonging to the Krox-20 subfamily indicates that they contain acidic regions at similar locations within their N-terminal region, suggesting that the functional organization of these proteins has been conserved during evolution.

The selectivity filter of a ligand-gated ion channel: The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation. Acetylcholine receptor; Ion channel; Selectivity filter; Molecular modeling

Growth of marine planktonic diatoms on inorganic and organic nitrogen

A study was conducted to determine if coastal diatoms from eutrophic waters (Werribee, Port Phillip Bay, Australia) are able to grow better than diatoms from oligotrophic waters (Bass Strait, Australia) on organic nitrogen compounds as their principal nitrogen sources. Eight clones of marine planktonic diatoms, belonging to 5 species (Skeletonema costatum, Asterionella japonica, Nitzschia closterium, Coscinodiscus sp., and Fragilaria sp.), were incubated with inorganic (either nitrate or ammonia) or organic (either urea, uric acid, alanine, aspartic acid, glutamic acid, glycine, serine, threonine, or valine) nitrogen sources and growth response was measured under high and low light intensities. All clones grew well on the organic as well as thorganic nitrogen compounds under both light regimes. Intraspecific differences were not great, as no appreciable difference was noted between clones from oligotrophic and eutrophic waters. The two negatively-charged amino acids, aspartic and glutamic acids, were somewhat less effective in supporting growth of some clones than were the other amino acids. Virtually all of the dissolved organic nitrogen (DON) compounds tested were utilizable for algal growth. Further, all clones appeared able to utilize at least some natural DON (uncharacterized) for cell division; in 1980, DON represented 97% of total nitrogen in Bass Strait and 83% of total nitrogen in Werribee waters. The results are consistent with previous findings on algal utilization of certain DON compounds and indicate comparable abilities of cells from oligotrophic and eutrophic coastal waters to assimilate these nutrients.