Homologous mutations on different subunits cause unequal but additive effects on n-alcohol block in the nicotinic receptor pore.

Hydrophobic antagonists of the nicotinic acetylcholine receptor inhibit channel activity by binding within the transmembrane pore formed by the second of four transmembrane domains (M2) on each of the receptor's subunits. Hydrophobic mutagenesis near the middle (10' locus) of the alpha-subunit M2 domain results in channels that are much more sensitive to block by long-chain alcohols and general anesthetics, indicating that the inhibitory site on wild-type receptors is nearby. To determine whether other receptor subunits also contribute to the blocker site, the hydrophobic mutagenesis strategy was extended to all four subunits at 10' loci. alpha S10'l causes the largest increase in apparent hexanol binding (4.3-fold compared to wild type), approximately twice the size of the change caused by beta T10'l (2.2-fold). gamma A10'l and delta A10'l mutations cause much smaller changes in apparent hexanol binding affinity (about 1.2-fold each), even when corrected for their smaller degree of side-chain hydrophobicity changes. When 10'l mutant subunits are coexpressed, the change from wild type in apparent hexanol binding energy (delta delta Gmixture) is roughly equal to the sum of hexanol binding energy changes for the constituent mutant subunits (sigma delta delta Gsubunits). The simplest model consistent with these results is one in which hydrophobic blockers make simultaneous contact with all five M2 10' residues, but the extent of contact is much greater for the alpha and beta than for gamma and delta side chains.     

Charges in the Cytoplasmic Pore Control Intrinsic Inward Rectification and Single-Channel Properties in Kir1.1 and Kir2.1 Channels

An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K⁺ with channel pore may be required for generating open-channel fluctuations in the E224G mutant.     

A hydrophobic photolabel inhibits nicotinic acetylcholine receptors via open-channel block following a slow step.

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) is a hydrophobic inhibitor of nicotinic acetylcholine receptors (nAChRs) and a photolabel that incorporates both at the lipid-protein interface and within the gated pore. On the basis of Torpedo vesicle studies, TID is thought to selectively inhibit the closed nAChR state. The nAChR site(s) mediating TID inhibition is unknown. We investigated the state dependence and kinetics of TID inhibition electrophysiologically using rapidly superfused membrane patches expressing mouse muscle nAChRs. Currents from patches simultaneously exposed to ACh and TID show no inhibition of peak currents relative to acetylcholine (ACh) alone but demonstrate slow (10 s(-1)) TID inhibition. Patch preexposure to TID before ACh results in a burst of current followed by rapid [TID]-dependent inhibition at a bimolecular rate of 1.8 x 10(8) M(-1) s(-1), indicating that TID selectively inhibits open channels. We also determined sensitivity to TID in two nAChRs containing mutations in their pore-forming M2 domains. The alphaL251T mutation eliminates sensitivity to TID inhibition, while the alphaS252I mutation enhances this sensitivity 4-fold compared to wild type. These results indicate that TID inhibition of nAChRs follows two distinct kinetic steps. The rate-limiting step, which shows features suggesting a diffusion barrier, precedes rapid open-state-dependent TID binding to an inhibition site near the putative nAChR gate.     

Molecular dynamics simulation of the M2 helices within the nicotinic acetylcholine receptor transmembrane domain: structure and collective motions

Multiple nanosecond duration molecular dynamics simulations were performed on the transmembrane region of the Torpedo nicotinic acetylcholine receptor embedded within a bilayer mimetic octane slab. The M2 helices and M2-M3 loop regions were free to move, whereas the outer (M1, M3, M4) helix bundle was backbone restrained. The M2 helices largely retain their hydrogen-bonding pattern throughout the simulation, with some distortions in the helical end and loop regions. All of the M2 helices exhibit bending motions, with the hinge point in the vicinity of the central hydrophobic gate region (corresponding to residues alphaL251 and alphaV255). The bending motions of the M2 helices lead to a degree of dynamic narrowing of the pore in the region of the proposed hydrophobic gate. Calculations of Born energy profiles for various structures along the simulation trajectory suggest that the conformations of the M2 bundle sampled correspond to a closed conformation of the channel. Principal components analyses of each of the M2 helices, and of the five-helix M2 bundle, reveal concerted motions that may be relevant to channel function. Normal mode analyses using the anisotropic network model reveal collective motions similar to those identified by principal components analyses.     

Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor

Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.     

Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.     

Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.     

Effects of Aliphatic Alcohols and Food Additives on Nicotinic Acetylcholine Receptors in Xenopus Oocytes

To study the effects of food additives on nicotinic acetylcholine receptors (nAChR), they were expressed in Xenopus oocytes that received an injection of mRNA prepared from electroplax of Electrophorus electricus. The response of nAChR elicited by acetylcholine (ACh) was measured electrophysiologically in the presence and absence of aliphatic alcohols and food additives. All compounds examined inhibited nAChR non-competitively in a concentration-dependent way. The inhibition was stronger when the inhibitors were perfused lmin before ACh, than when they were perfused simultaneously with ACh. The inhibition of nAChR by aliphatic alcohols (propanol to hexanol) increased as the number of carbon chains increased. The addition of alcohols and food additives did not affect the desensitization of nAChR caused by 2 μm ACh. These results suggest that alcohols and food additives bind to the anesthetic binding site in nAChR and inhibit it noncompetitively. However, these compounds will not hinder signal transmission in neuromuscular junctions under physiological conditions, because their inhibition constants are more than 1 mm and muscles usually have more receptors than the number necessary for signal transmission.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.     

Amine blockers of the cytoplasmic mouth of sodium channels: a small structural change can abolish voltage dependence.

Many drugs block sodium channels from the cytoplasmic end (Moczydlowski, E., A. Uehara, X, Guo, and J. Heiny. 1986. Isochannels and blocking modes of voltage-dependent sodium channels. Ann. N.Y. Acad. Sci. 479:269-292.). Lidocaine, applied to either side of the membrane, induces two blocking modes, a rapid, voltage-dependent open-channel block, and a block of the inactivated channel that occurs on a 1000-fold slower timescale. Here we describe the actions of several lidocaine-related amines on batrachotoxin(BTX)-activated bovine cardiac sodium channels incorporated into planar lipid bilayers. We applied blocking amines from the intracellular side and examined the structural determinants of fast, open-channel block. Neither hydroxyl nor carbonyl groups, present in the aryl-amine link of lidocaine, were necessary, indicating that hydrogen bonding between structures in the aryl-amine link and the channel is not required. Block, however, was significantly enhanced by addition of an aromatic ring, or by the lengthening of aliphatic side chains, suggesting that a hydrophobic domain strengthens binding while the amine group blocks the pore. For most blockers, depolarizing potentials enhanced block, with the charged amine group apparently traversing 45-60% of the transmembrane voltage. By contrast, block by phenylhydrazine was essentially voltage-independent. The relatively rigid planar structure of phenylhydrazine may prevent the charged amino end from entering the electric field when the aromatic ring is bound. The relation between structural features of different blockers and their sensitivity to voltage suggests that the transmembrane voltage drops completely over less than 5 A. We raise the possibility that the proposed hydrophobic binding domain overlaps the endogenous receptor for the inactivation gate. If so, our data place limits on the distance between this receptor and the intrapore site at which charged amines bind.     

Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships

GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundstr?m, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations.     

Architecture of the Neuronal Nicotinic Acetylcholine Receptor Ion Channel at the Binding Site of bis-Ammonium Blockers

Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively. Received: 6 September 1995/Revised: 12 March 1996     

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.     

Interfacial and distal-heme pocket mutations exhibit additive effects on the structure and function of hemoglobin

Protein engineering strategies seek to develop a hemoglobin-based oxygen carrier with optimized functional properties, including (i) an appropriate O 2 affinity, (ii) high cooperativity, (iii) limited NO reactivity, and (iv) a diminished rate of auto-oxidation. The mutations alphaL29F, alphaL29W, alphaV96W and betaN108K individually impart some of these traits and in combinations produce hemoglobin molecules with interesting ligand-binding and allosteric properties. Studies of the ligand-binding properties and solution structures of single and multiple mutants have been performed. The aromatic side chains placed in the distal-heme pocket environment affect the intrinsic ligand-binding properties of the mutated subunit itself, beyond what can be explained by allostery, and these changes are accompanied by local structural perturbations. In contrast, hemoglobins with mutations in the alpha 1beta 1 and alpha 1beta 2 interfaces display functional properties of both "R"- and "T"-state tetramers because the equilibrium between quaternary states is altered. These mutations are accompanied by global structural perturbations, suggesting an indirect, allostery-driven cause for their effects. Combinations of the distal-heme pocket and interfacial mutations exhibit additive effects in both structural and functional properties, contribute to our understanding of allostery, and advance protein-engineering methods for manipulating the O 2 binding properties of the hemoglobin molecule.     

Mechanism-based approach to the successful prevention of cocaine inhibition of the neuronal (alpha 3 beta 4) nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a family of five channel-forming proteins that regulate communication between the approximately 10(12) cells of the nervous system. A minimum mechanism of inhibition of the muscle-type nAChR (1) by the noncompetitive inhibitors cocaine and MK-801 [(+)-dizocilpine, an anticonvulsant] indicated they bind to a regulatory site, with higher affinity for the closed-channel form than for the open-channel form, thus shifting the equilibrium toward the closed-channel form and inhibiting receptor function. The mechanism predicts that compounds that bind to this regulatory site with equal or higher affinity for the open-channel conformation than for the closed-channel conformation will prevent receptor inhibition (1). Does a neuronal form of the receptor behave similarly? The mechanism of inhibition of the neuronal nAChR by cocaine and MK-801 using rapid chemical kinetic techniques was investigated. The alpha3beta4 nAChR stably expressed in HEK 293 cells was used in these investigations. Whole-cell currents originated from a major and minor nAChR isoform. Only the major isoform has been characterized. For the dominant, rapidly desensitizing isoform, the carbamoylcholine dissociation constant for the site controlling receptor activation, Kd, is 2 mM; the channel-opening equilibrium constant, Phi(-1), is 4; and the dominant desensitization rate constant, k34, is 20 s(-1). Cocaine inhibits the receptor noncompetitively, with an apparent KI of 84 and 26 microM at high and low carbamoylcholine concentrations, at which concentrations the receptor is mainly in the open- or closed-channel form, respectively. Similar results were obtained with MK-801. A combinatorially synthesized RNA ligand and a cocaine analogue alleviated cocaine inhibition of this neuronal receptor.     

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.     

Differential effects of ethanol and hexanol on the Escherichia coli cell envelope.

Both ethanol and hexanol inhibited the growth of Escherichia coli, but their effects on the organization and composition of the cell envelope were quite different. Hexanol (7.8 x 10(-3) mM) increased membrane fluidity, whereas ethanol (0.67 M) had little effect. During growth in the presence of ethanol, the proportion of unsaturated fatty acids increased. The opposite change was induced by hexanol. Unlike hexanol, growth in the presence of ethanol resulted in the production of un-cross-linked peptidoglycan with subsequent lysis. Salt (0.3 M) protected cells against ethanol-induced lysis but potentiated growth inhibition by hexanol. Mutants isolated for resistance to ethanol-induced lysis synthesized cross-linked peptidoglycan during growth in the presence of ethanol but remained sensitive to hexanol. A general hypothesis was presented to explain the differential effects of ethanol and hexanol. All alcohols are viewed as similar in having both an apolar chain capable of interacting with hydrophobic environments and a hydroxyl function capable of hydrogen bonding. The differential effects of short-chain alcohols may represent effects due to the high molar concentrations of hydrogen bonding groups with an apolar end within the environment. These may replace bound water in some cases. With longer-chain alcohols such as hexanol, the effects of the acyl chain would dominate, and limitations of solubility and cellular integrity would mask these hydroxyl effects.     

Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel

Dendrotoxin proteins isolated from Mamba snake venom block potassium channels with a high degree of specificity and selectivity. Using site-directed mutagenesis we have identified residues that constitute the functional interaction surfaces of delta-dendrotoxin and its voltage-gated potassium channel receptor. delta-Dendrotoxin uses a triangular patch formed by seven side-chains (Lys3, Tyr4, Lys6, Leu7, Pro8, Arg10, Lys26) to block K(+) currents carried by a Shaker potassium channel variant. The inhibitory surface of the toxin interacts with channel residues at Shaker positions 423, 425, 427, 431, and 449 near the pore. Amino acid mutations that interact across the toxin-channel interface were identified by mutant cycle analysis. These results constrain the possible orientation of dendrotoxin with respect to the K(+) channel structure. We propose that dendrotoxin binds near the pore entryway but does not act as a physical plug.