methanethiosulfonate and (2-(trimethylammonium)ethyl)methanethiosulfonate

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.

Topics: Amino Acid Sequence; Binding Sites; Biological Transport; Biotinylation; Cell Line; Cell Membrane; Chlorine; Creatine; Cysteine; Dose-Response Relationship, Drug; Humans; Ions; Membrane Transport Proteins; Mesylates; Models, Biological; Molecular Sequence Data; Mutagenesis; Mutation; Protein Binding; Protein Structure, Tertiary; Sodium; Solvents; Sulfhydryl Reagents; Time Factors; Transfection

The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

Topics: Animals; Anions; Arginine; Cysteine; Cystic Fibrosis Transmembrane Conductance Regulator; Disulfides; Electric Conductivity; Ethyl Methanesulfonate; Female; Humans; Hydrogen-Ion Concentration; Lysine; Membrane Potentials; Mercaptoethanol; Mesylates; Models, Biological; Oocytes; Patch-Clamp Techniques; Perfusion; Xenopus

The structure of the pore region of the alpha subunit of the bovine rod cyclic nucleotide-gated channel was probed using cysteine-scanning mutagenesis and hydrophilic sulfhydryl-reactive methanethiosulfonate (MTS) reagents. A region homologous to the pore helix in the X-ray crystal structure of the KcsA K(+) channel showed a helical pattern of reactivity with externally applied MTS reagents. Surprisingly, three out of four of the reactive residues, all on one face of the pore helix, only reacted with MTS reagents in the closed state. A residue on the opposite face of the helix only reacted with MTS reagents in the open state. These results indicate that the pore helix (or its surroundings) undergoes a change in conformation, perhaps involving a rotation around its long axis, that opens a gate localized to the selectivity filter of the channel.

Topics: Animals; Bacterial Proteins; Cattle; Cells, Cultured; Cyclic Nucleotide-Gated Cation Channels; Ethyl Methanesulfonate; Indicators and Reagents; Ion Channel Gating; Ion Channels; Mesylates; Models, Molecular; Mutagenesis, Site-Directed; Oocytes; Patch-Clamp Techniques; Potassium Channels; Protein Conformation; Protein Structure, Secondary; Retinal Rod Photoreceptor Cells; Sequence Homology, Amino Acid; Sulfhydryl Reagents; Water; Xenopus

To understand the wide variation of calcium permeability seen in native and recombinant 5-HT3 receptor (5-HT3R) channels, we reported previously the novel hypothesis that the serotonin 5-HT3R subunit can co-assemble with the alpha4 subunit of the nicotinic acetylcholine receptor (van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. & Vijverberg, H. P. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11456-11461). To test the hypothesis that the alpha4 subunit contributes to the lining of the pore of the resulting 5-HT3R channel, a mutant nicotinic alpha4 subunit with a reactive cysteine residue engineered into the putative pore region was constructed by substituting the leucine at position 285 (alpha4-L285C). The sulfhydryl-modifying reagent [2-(trimethylammonium) ethyl]methanethiosulfonate (MTSET) reduced the acetylcholine-induced current in oocytes expressing this mutant nicotinic alpha4-L285C subunit along with the nicotinic beta2 subunit by approximately 60%. When the alpha4-L285C subunit was co-expressed with the 5-HT3R subunit, both MTSET and silver nitrate (AgNO3), another cysteine-modifying reagent, significantly reduced the serotonin-induced current. No reduction was seen when the 5-HT3R was expressed alone or with the wild-type alpha4 subunit. These data provide direct molecular evidence that the nicotinic alpha4 subunit co-assembles with the 5-HT3R subunit and forms an integral part of the ion channel pore.

Topics: Animals; Cysteine; Genetic Engineering; Indicators and Reagents; Mesylates; Oocytes; Receptors, Nicotinic; Receptors, Serotonin; Receptors, Serotonin, 5-HT3; Silver Nitrate; Xenopus