We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtUA gene, which encodes the mannitol-specffic enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtL4 were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N-and C-terminal domains. This phospho-exchange reaction (transphosphorylation) has been shown to occur vectorially in whole cells and membrane vesicles (35,36).The mannitol permease has been purified (11) and extensively characterized (7,10,30,32) been cloned and sequenced (17). These studies have revealed that the protein is made up of a single kind of polypeptide chain consisting of 637 amino acid residues (11, 17) that is highly specific for D-mannitol (12, 19) and may function as a dimer in the membrane (24,30,33,41). The topography of the mannitol permease with respect to the membrane has also been studied, and it is clear that this protein consists of a hydrophobic, membrane-bound Nterminal domain (residues 1 to 334) and a hydrophilic Cterminal domain (residues 335 to 637) that is exposed on the cytoplasmic surface of the inner membrane (9,17,42). In addition to carrying out the transport and phosphorylation functions described above, the mannitol permease functions as the primary chemotactic receptor for D-mannitol in E. coli (20).The domain structure of the mannitol permease and its multiple functions suggest...

To identify the mechanisms underlying the faster activation kinetics in Kv1.2 channels compared to Kv2.1 channels, ionic and gating currents were studied in rat Kv1.2 and human Kv2.1 channels heterologously expressed in mammalian cells. At all voltages the time course of the ionic currents could be described by an initial sigmoidal and a subsequent exponential component and both components were faster in Kv1.2 than in Kv2.1 channels. In Kv1.2 channels, the activation time course was more sigmoid at more depolarized potentials, whereas in Kv2.1 channels it was somewhat less sigmoid at more depolarized potentials. In contrast to the ionic currents, the ON gating currents were similarly fast for both channels. The main portion of the measured ON gating charge moved before the ionic currents were activated. The equivalent gating charge of Kv1.2 ionic currents was twice that of Kv2.1 ionic currents, whereas that of Kv1.2 ON gating currents was smaller than that of Kv2.1 ON gating currents. In conclusion, the different activation kinetics of Kv1.2 and Kv2.1 channels are caused by rate-limiting reactions that follow the charge movement recorded from the gating currents. In Kv1.2 channels, the reaction coupling the voltage-sensor movement to the pore opening contributes to rate limitation in a voltage-dependent fashion, whereas in Kv2.1 channels, activation is additionally rate-limited by a slow reaction in the subunit gating.

The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the beta-methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly74 of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.

Depolarizing voltage steps activate voltage-dependent K(+) (Kv) channels by moving the voltage sensor, which triggers a coupling reaction leading to the opening of the pore. We constructed chimeric channels in which intracellular regions of slowly activating Kv2.1 channels were replaced by respective regions of rapidly activating Kv1.2 channels. Substitution of either the N-terminus, S4-S5 linker, or C-terminus generated chimeric Kv2.1/1.2 channels with a paradoxically slow and approximately exponential activation time course consisting of a fast and a slow component. Using combined chimeras, each of these Kv1.2 regions further slowed activation at the voltage of 0 mV, irrespective of the nature of the other two regions, whereas at the voltage of 40 mV both slowing and accelerating effects were observed. These results suggest voltage-dependent interactions of the three intracellular regions. This observation was quantified by double-mutant cycle analysis. It is concluded that interactions between N-terminus, S4-S5 linker, and/or C-terminus modulate the activation time course of Kv2.1 channels and that part of these interactions is voltage dependent.

The α subunits of CNG channels of retinal photoreceptors (rod) and olfactory neurons (olf) are proteins that consist of a cytoplasmic NH2 terminus, a transmembrane core region (including the segments S1–S6), and a cytoplasmic COOH terminus. The COOH terminus contains a cyclic nucleotide monophosphate binding domain NBD) that is linked by the C-linker (CL) to the core region. The binding of cyclic nucleotides to the NBD promotes channel opening by an allosteric mechanism. We examined why the sensitivity to cGMP is 22 times higher in olf than in rod by constructing chimeric channels and determining the [cGMP] causing half maximum channel activity (EC50). The characteristic difference in the EC50 value between rod and olf was introduced by the NH2 terminus and the core-CL region, whereas the NBD showed a paradoxical effect. The difference of the free energy difference Δ(ΔG) was determined for each of these three regions with all possible combinations of the other two regions. For rod regions with respect to corresponding olf regions, the open channel conformation was destabilized by the NH2 terminus (Δ(ΔG) = −1.0 to −2.0 RT) and the core-CL region (Δ(ΔG) = −2.0 to −2.9 RT), whereas it was stabilized by the NBD (Δ(ΔG) = 0.3 to 1.1 RT). The NH2 terminus deletion mutants of rod and olf differed by Δ(ΔG) of only 0.9 RT, whereas the wild-type channels differed by the much larger value of 3.1 RT. The results show that in rod and olf, the NH2 terminus, the core-CL region, and the NBD differ by characteristic Δ(ΔG) values that do not depend on the specific composition of the other two regions and that the NH2 terminus generates the main portion of Δ(ΔG) between the wild-type channels.