The structural bases of general anesthetic action on a neuronal K ؉ channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and sitedirected mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K ؉ channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.General anesthetics (including 1-alkanols) at clinically relevant concentrations mainly alter the function of neuronal ion channels by a direct action (1-5). Site-directed mutagenesis has provided strong evidence favoring the presence of critical protein sites necessary for the interaction between 1-alkanols and the ion channel protein (e.g. Refs. 6 -12). Furthermore, these studies suggest that diverse sites but similar molecular mechanisms may underlie general anesthesia and alcohol intoxication. The application of mutational analysis and electrophysiology to the investigation of model ion channels that directly interact with 1-alkanols can provide detailed information about the general characteristics of the 1-alkanol sites in membrane proteins and the molecular interactions that underlie the effects of general anesthetics and 1-alkanols on more complex neuronal ion channels.We have investigated the molecular properties of the putative 1-alkanol site in Drosophila dShaw2 K ϩ channels. These channels are expressed in Drosophila neurons and exhibit low voltage sensitivity, low open probability, and no inactivation (13-16). dShaw2 channels are selectively inhibited by anesthetic concentrations of ethanol (25-100 mM) and homologous 1-alkanols in a manner that agrees with a drug-receptor interaction (6,17). This inhibition is due to the stabilization of the closed state(s) of the channel by preferentially reducing the probability of entering a long duration open state (with no effect on the mean open time) (6). The earlier work also showed that dShaw2 subunits confer 1-alkanol sensitivity to hybrid channels and suggested that...

This study examined the emetic activity of several staphylococcal enterotoxin type A and B (SEA and SEB, respectively) mutants that had either one or two amino acid residue substitutions. New sea gene mutations were constructed by site-directed mutagenesis; gene products were obtained with glycine residues at position 25, 47, 48, 81, 85, or 86 of mature SEA. Culture supernatants from Staphylococcus aureus RN4220, or derivatives containing either sea or a sea mutation, were analyzed for the ability to stimulate proliferation of murine splenocytes, as determined by incorporation of [3H]thymidine. Culture supernatants containing SEA-N25G (a SEA mutant with a substitution of glycine for the asparagine residue at position 25), SEA-F47G, or SEA-L48G did not stimulate T-cell proliferation, unlike supernatants containing the other substitution mutants. Purified preparations of SEA-N25G had weak activity and those of SEA-F47G and SEA-L48G had essentially no activity in the T-cell proliferation assay. All mutants except SEA-V85G, which was degraded by monkey stomach lavage fluid in vitro, were tested for emetic activity. SEA-C106A and two SEB mutants, SEB-D9N/N23D and SEB-F44S (previously referred to as BR-257 and BR-358, respectively), whose construction and altered immunological properties have been reported previously, were also tested in the emetic assay. Each mutant was initially administered intragastrically at doses of 75 to 100 micrograms per animal; if none of the animals responded, the dose was increased four-to fivefold. SEA-F47G, SEA-C106A, and SEB-D9N/N23D were the only mutants that did not induce vomiting at either dose tested; these three mutants had reduced immunological activity. However, there was not a perfect correlation between immunological and emetic activities; SEA-L48G and SEB-F44S retained emetic activity, although they had essentially no T-cell-stimulatory activity. These studies suggest that these two activities can be dissociated.

Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K + channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 μs) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K + channels in Xenopus oocyte inside-out patches. As expected for a secondorder mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only ∼2-fold between ethanol and 1-hexanol), the secondorder association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K + channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the α-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.Alcohol and general anesthetic agents interact with related and relatively specific binding sites in multiple protein targets (1-6). Aliphatic alcohols (e.g., 1-alkanols) have been used to probe the physicochemical properties of these binding sites (7-11). The results of these studies are consistent with the presence of physically circumscribed hydrophobic protein cavities that constitute the alcohol and general anesthetic sites. Also, studies with soluble model proteins have examined the relationship between anesthetic solubility and anesthetic action along with structure-function analysis and thermodynamic arguments and suggest that polar interactions also contribute to the binding of alcohol and inhaled anesthetics (5,9,(12)(13)(14)(15). Polar interactions are also likely to contribute to the binding of general anesthetic agents to ion channels, which are critical physiological targets of these agents (including 1-alkanols). Our earlier work has shown that the Drosophila Shaw2 K + channel is a robust model for investigating the proteinbased theories of alcohol intoxication and general anesthetic action (16)(17)(18) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript channels are members of the superfamily of voltage-gated K + channels (Kv channels)....

Harris, Thanawath, Andrew R. Graber, and Manuel Covarrubias. Allosteric modulation of a neuronal K ϩ channel by 1-alkanols is linked to a key residue in the activation gate. Am J Physiol Cell Physiol 285: C788-C796, 2003; 10.1152/ajpcell.00113.2003.-The selective inhibition of neuronal Shaw2 K ϩ channels by 1-alkanols is conferred by the internal S4-S5 loop, a region that also contributes to the gating of voltage-gated K ϩ channels. Here, we applied alanine scanning mutagenesis to examine the contribution of the S5 and S6 segments to the allosteric modulation of Shaw2 K ϩ channels by 1-alkanols. The internal section of S6 is the main activation gate of K ϩ channels. While several mutations in S5 and S6 modulated the inhibition of the channels by 1-butanol and others had no effect, a single mutation at a key site in S6 (P410A) converted this inhibition into a dramatic dose-dependent potentiation (ϳ2-fold at 15 mM and ϳ6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant ␣-helix kink. The P410A currents in the presence of 1-butanol also exhibited novel kinetics (faster activation and slow inactivation). Internal application of 15 mM 1-butanol to inside-out patches expressing P410A did not significantly affect the mean unitary currents (ϳ2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (ϳ2-to 4-fold). All effects displayed a fast onset and were fully reversible upon washout. The results suggest that the allosteric modulation of the Shaw2 K ϩ channel by 1-alkanols depends on a critical link between the PVP motif and activation gating. This study establishes the Shaw2 K ϩ channel as a robust model to investigate the mechanisms of alcohol intoxication and general anesthesia. alcohol; anesthesia; gating; scanning mutagenesis; Shaw channels ALIPHATIC ALCOHOLS (1-alkanols) and general anesthetics directly interact with critical sites in neuronal ion channels and, thereby, alter their function (7,28,29,34,36). Whereas in some instances ion channel function is inhibited, in others it is potentiated. Such effects could result from pore blockade or altered gating and are thought to constitute the physiological basis of acute alcohol intoxication and general anesthesia. However, little is known about the structural basis of these interactions and how they may change gating of ion channels. A current hypothesis proposes that 1-alkanols and general anesthetics bind to amphiphilic protein-protein interfaces involved in the conformational changes that underlie channel gating (2, 10, 14, 28). We have tested this hypothesis by examining the role of regions that contribute to the putative gating machinery of voltage-gated K ϩ channels (Kv channels). The neuronal Shaw2 K ϩ channel from Drosophila melanogaster is suitable for these studies because it is selectively inhibited by pharmacologically relevant concentrations of alcohol (3, 4). Shaw2 K ϩ channels are also special members of the superfamily of Kv channels becau...

Mycophenolic acid (MPA) is a potent and specific inhibitor of mammalian inosine-monophosphate dehydrogenases (IMPDH); most microbial IMPDHs are not sensitive to MPA. MPA-resistant mutants of human IMPDH type II were isolated in order to identify the structural features that determine the species selectivity of MPA. Three mutant IMPDHs were identified with decreased affinity for MPA. The mutation of Gln 277 3 Arg causes a 9-fold increase in the K i of MPA, a 5-6-fold increase in the K m values for IMP and NAD, and a 3-fold decrease in k cat relative to wild type. The mutation of Ala 462 3 Thr causes a 3-fold increase in the K i for MPA, a 2.5-fold increase in the K m for NAD, and a 1.5-fold increase in k cat . The combination of these two mutations does not increase the K i for MPA, but does increase the K m for NAD 3-fold relative to Q277R and restores k cat to wild type levels. Q277R/A462T is the first human IMPDH mutant with increased K i for MPA and wild type activity. The third mutant IMPDH contains two mutations, Phe 465 3 Ser and Asp 470 3 Gly. K i for MPA is increased 3-fold in this mutant enzyme, and K m for IMP is also increased 3-fold, while the K m for NAD and k cat are unchanged. Thus increases in the K i for MPA do not correlate with changes in K m for either IMP or NAD, nor to changes in k cat . All four of these mutations are in regions of the IMPDH that differ in mammalian and microbial enzymes, and thus can be structural determinants of MPA selectivity. Inosine-monophosphate dehydrogenase (IMPDH)1 catalyzes the oxidation of IMP to XMP with the concomitant conversion of NAD to NADH (Fig. 1). This reaction is the rate-limiting step in guanine nucleotide biosynthesis, and rapidly growing cells have increased levels of IMPDH (1). Inhibitors of IMPDH have antiproliferative activity and are used clinically for cancer, viral, and immunosuppressive chemotherapy (2-4). Moreover, differences in the properties of microbial and mammalian IMPDHs suggest that species-selective IMPDH inhibitors can be designed, which will be useful for anti-infective chemotherapy (5-7). Two human IMPDH isozymes exist; type I is constitutively expressed, while type II is expressed in rapidly proliferating cells (8 -11). The IMPDH reaction involves attack of Cys 331 (human type II numbering) at the 2-position of IMP, followed by expulsion of the hydride to NAD (Fig. 1) (Fig. 1), and the crystal structure of the E-XMP⅐MPA complex of IMPDH from Chinese hamster has recently been solved (12,19,20 Random mutagenesis followed by selection for the ability to grow in the presence of MPA can identify mutations in IMPDH that confer MPA resistance. Selection for MPA resistance has previously been reported in both mammalian and parasite systems. In most cases MPA resistance resulted from increased expression of IMPDH, usually via gene amplification (24 -26). Mutant IMPDHs with altered sensitivity to MPA have been reported in murine lymphoma and leukemia cells, although identity of these mutations and their effect on enzyme activity were no...