Structural locus of the pH gate in the Kir1.1 inward rectifier channel.

The closed-state crystal structure of prokaryotic inward rectifier, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward rectifier, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.

Structure-based design of non-peptide HIV protease inhibitors.

A number of structurally novel P2-ligands have been designed and synthesized. Incorporation of these ligands in the (R)-(hydroxyethyl)sulfonamide isostere provided a series of potent non-peptidyl HIV protease inhibitors.

Modifying the temporal profile of the high-potency sweetener neotame.

It is possible, using hydrophobic organic acids (such as cinnamate) or hydroxyamino acids (such as serine and tyrosine), to modify the temporal profile of the high-potency sweetener neotame. On the basis of Monte Carlo simulations, it was concluded that it is unlikely that this effect is due to direct interaction between the neotame molecule and the taste modifier. It is shown, using conformational analysis and molecular modeling, that the taste modifiers can adopt low-energy conformers which mimic the proposed active conformation of neotame, which suggests that the modifiers may compete for binding at the receptor site.

Regulation of ROMK by extracellular cations.

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (K(ir)1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10 mM increased whole-cell outward conductance by a factor of 3.4 +/- 0.4 in 15 min and by a factor of 5.7 +/- 0.9 in 30 min (n = 22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5 +/- 0.5 over a period of 40 min (n = 15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10 mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4 +/- 0.8 pS to 11.1 +/- 0.8 pS (10 mM K, n = 8) or 11.7 +/- 1.2 pS (Cs, n = 8). There was no effect of either 10 mM K or cesium on the high open probability (P(o) = 0.97 +/- 0.01; n = 12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50 mM K. In cell-attached patches, exposure to 50 mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50 mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (K(ir)2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10 mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway.

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.

Detection of Trypanosoma congolense antibodies with indirect ELISAs using antigen-precoated microtitre plates.

The study reports the performance of four indirect enzyme-linked immunosorbent assays (ELISAs) for antibody (AB) detection using microtitre plates which were precoated with native or heat/detergent denatured antigens (AGs) from Trypanosoma congolense (T.c.) and T. vivax (T.v.), and stored for between 1 to 206 days at +37 degrees C. Bovine serum samples were obtained by sequential bleeding of 3-months old T.c.-infected bulls and their uninfected cohorts, as well as by a single bleeding of uninfected adult cattle. The first day of AB detection, and observations on samples after this (defined as estimated ELISA sensitivity), depended on the cut-off value in the specific ELISAs. Cut-off values from pre- and early post-infection samples of individual animals demonstrated a seroconversion in all ELISAs on average after 10-15 days post-infection (dpi). The AB detection was delayed in the T.c. native and denatured AG-based ELISAs using cut-off points from uninfected cohort cattle (16.5 dpi, 19.3 dpi) and the adult cattle population (22.1 dpi, 25.0 dpi). The T.v. AG-based ELISAs however lacked crossreactiviy to T.c. ABs. The estimated sensitivity of each T.c. AG-based ELISA was above 96% throughout, but significantly lower for the T.c. native AG-based ELISA (91.1%) when the adult cattle derived cut-off point was used (p<0.01). The sensitivity of the phase contrast buffy coat technique was similar to the T.c. AG-based ELISAs, but significantly lower when the T.c. denatured AG-based ELISA was used at the adult cattle derived cut-off point (p<0.05). The implications of the results and future research aspects on ELISAs to detect trypanosomal ABs and AGs are discussed.

Active conformations of neotame and other high-potency sweeteners.

We carried out extensive conformational analysis of three high-potency sweeteners: neotame, superaspartame, and SC-45647. We then identified six possible pharmacophore features (carboxylate, two hydrophobic groups, and three NH groups) and wrote a computer program to exhaustively compare intramolecular distances among all possible sets of five-point pharmacophores (carboxylate + two hydrophobic groups + two NH groups) for the three compounds. The best pharmacophore model superimposes low-energy conformers of the three compounds in such a way that the five pharmacophore points match well both sterically and with respect to orientation of hydrogen bond donors and acceptors.

The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function.

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.

Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins.

Experiments have been conducted to determine the oligomeric state of the mitochondrial citrate transport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type and cysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity of the solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, a high specific transport activity is obtained with both the wild-type and Cys-less CTPs, thereby demonstrating the structural and functional integrity of the preparations. Two independent approaches were utilized to determine native molecular weight. First, CTP molecular weight was determined via nondenaturing size-exclusion chromatography. With this methodology we obtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs, respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a low concentration of the negatively charged detergent sarkosyl, which served to both impart a charge shift to the CTP and the protein standards, as well as to promote protein solubility. Via the second method, we obtained molecular weight values of 69,122 and 74,911 for the wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that following solubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore, disulfide bonds are not required for either dimer formation or stabilization. The dimeric state of the CTP has important implications for the structural basis underlying the CTP translocation mechanism.

The active site of pyrophosphate-dependent phosphofructo-1-kinase based on site-directed mutagenesis and molecular modeling.

Despite a low level of overall sequence identity between PPi-dependent and ATP-dependent phosphofructo-1-kinases (PFKs), similarities in active-site residues permit a convincing amino acid alignment of these two groups of kinases. Employing recent protein sequence and site-directed mutagenesis data along with the known three-dimensional coordinates of Escherichia coli ATP-dependent PFK, a model of the active site of PPi-dependent PFK was proposed. In addition to providing compatible placement of residues shown to be important by earlier mutagenesis studies, the model predicted an important role for two arginyl residues that are conserved in all known PPi-PFK sequences. Replacement by site-directed mutagenesis of these two residues with neutral amino acids in the PPi-PFK of Naegleria fowleri resulted in a substantial reduction in kcat while not altering the global structure of the enzyme. While the data indicate many similarities in the active-site structures and mechanisms of ATP-dependent and PPi-dependent PFKs, subtle differences, such as the relative roles of Arg residues in the active sites, have evolved in the development of these two subgroups of the PFK family.

Structure based design: novel spirocyclic ethers as nonpeptidal P2-ligands for HIV protease inhibitors.

A series of novel spirocyclic ethers were designed to function as nonpeptidal P2-ligands for HIV-1 protease inhibitors. Incorporation of designed ligands in the (R)-(hydroxyethylamino)sulfonamide isostere afforded potent HIV protease inhibitors.

Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere.

Design and synthesis of a series of very potent nonpeptide HIV protease inhibitors are described. The inhibitors are derived from novel high affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere.

Up-regulation of dopamine D1-receptors in the brain of 28-day-old rats exposed to the delta (delta) opioid agonist SNC80 during the preweaning period.

Twenty-eight-day-old rats exposed to the delta (delta) opioid receptor agonist SNC80 during the preweaning period exhibited a significant increase in the density and apparent dissociation constant of striatal dopamine D1-receptors. There were no significant effects on the binding characteristics of striatal D2-receptors or on D1- or D2-receptors in the nucleus accumbens. The results suggest that delta-opioid receptor mechanisms might be involved in certain neurological changes observed in offspring of mother addicted to opioids during nursing.

Stimulating dopamine D1 receptors increases the locomotor activity of developing rats.

To determine the role of dopamine D1 receptors in the locomotor activity of developing rats, male offspring were habituated to an animal activity monitor and were then injected with the dopamine D1 receptor antagonist, SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl- 2,3,4,5-tetrahydro-(1 H)-3-benzazepine), or vehicle and returned to the activity monitors. 30 min later, they were injected with the dopamine D1 receptor agonist, SKF 38393 (R(+)-1-phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol), or vehicle and were again placed in the activity boxes where their locomotor activity was monitored individually for 1 h. The litter was used as the unit for statistical analyses. There was a significant increase in the locomotor activity of 10- and 21-day-old offspring injected with SKF 38393. This effect was antagonized by pretreatment with SCH 23390. These data provide the strongest evidence to date that stimulation of dopamine D1 receptors increases the locomotor activity of habituated developing rats.

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.