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A Karlin

Publications and source records attributed to A Karlin.

At least 19 recordsLinked to original sources
PubMed1998

Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.

We have applied the substituted-cysteine-accessibility method (SCAM) to the M2 segment and the M1-M2 loop of the acetylcholine (ACh) receptor beta subunit. Each residue from beta P248 to beta D273 was mutated one at a time to Cys, and the mutant beta subunits were expressed together with wild-type alpha, beta, and delta subunits in Xenopus oocytes. For each of the mutants, the ACh-induced current was near wild-type. The accessibility of the substituted Cys was inferred from the irreversible inhibition or potentiation of ACh-induced current by methanethiosulfonate (MTS) derivatives added extracellularly. Inhibition by MTSethylammonium of beta G255C, in the narrow part of the channel, was mainly due to a reduction in the single-channel conductance. Conversely, potentiation by MTSethylammonium of beta V266C, in a wider part of the channel, was mainly due to an increase in channel open-time. Two substituted Cys at the intracellular end of M2 and three at the extracellular end were accessible to MTSethylammonium in the absence of ACh. Three additional Cys in the middle of M2 and three in the M1-M2 loop were accessible in the presence of ACh. In the presence of ACh, the secondary structure of beta M2 is alpha-helical from beta G255 to beta V266 and extended from beta L268 to beta D273. The accessible residues in beta M2 are remarkably hydrophobic, while the accessible residues in the M1-M2 loop are charged. beta M2, like alpha M2, alpha M1, and beta M1, undergoes widespread structural changes concomitant with gating, but the gate itself is close to the intracellular end of the channel. Many aligned residues in the M2 segments of alpha and beta are not identically accessible, indicating that the two subunits contribute differently to the channel lining.

Acetylcholine↗
PubMed1998

The location of the gate in the acetylcholine receptor channel.

The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, alphaS239 to alphaT244, in the alpha subunit M1-M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between alphaG240 and alphaT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues alphaG240, alphaE241, alphaK242, and alphaT244 line a narrow part of the channel, in which this gate is located.

Acetylcholine↗
PubMed1998

State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the alpha subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for alpha Thr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with alpha T244C, alpha L251C, and alpha L258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of alpha M2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than alpha Thr244.

Algorithms↗
PubMed1998

Delimiting the binding site for quaternary ammonium lidocaine derivatives in the acetylcholine receptor channel.

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle alpha subunit were mutated one at a time to cysteine and expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from alphaVal255, one third of the distance in from the extracellular end of M2, to alphaGlu241, flanking the intracellular end of M2, but not with cysteine substituted for alphaLeu258 or alphaGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for alphaLeu258 and alphaGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect alphaL258C and alphaE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-A overall length of the blockers and the alpha-helical conformation of M2 in the open state, the binding site for both blockers extends from alphaVal255 down to alphaSer248.

Amino Acid Sequence↗
PubMed1997

Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit.

The substituted cysteine accessibility method (SCAM) was applied to the first membrane-spanning segment (M1) of the mouse-muscle acetylcholine (ACh) receptor beta subunit. One at a time, each residue from betaR219 to betaP247, except betaC233, was mutated to Cys, and the mutant beta subunits were expressed together with wild-type alpha, gamma, and delta in Xenopus oocytes. All 28 mutants yielded functional receptors. The accessibility of the substituted Cys to the methanethiosulfonate (MTS) derivatives, MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), and MTS ethylsulfonate (MTSES), added extracellularly in the absence or the presence of ACh, was inferred from their irreversible effects on ACh-induced current. Three consecutive residues close to the extracellular end of M1, betaF224C, betaY225C, and betaL226C, reacted both in the absence and presence of ACh, and one deeper residue, betaV229C reacted only in the presence of ACh. betaV229C also reacted with 2-aminoethyl-2-aminoethanethiosulfonate (AEAETS) and with 2-hydroxyethyl MTS (MTSEH). The rate constants for the reactions of betaV229C with MTSEA, which permeates the open channel, and with MTSEH, which is uncharged, were independent of membrane potential. The rate constant for the reaction of the doubly positively charged AEAETS, however, was dependent on membrane potential, consistent with the exposure of betaV229C in the open channel. The N-terminal third of betaM1, like that of alphaM1, contributes to the lining of the channel and undergoes structural changes during gating.

Animals↗
PubMed1997

Functional effects on the acetylcholine receptor of multiple mutations of gamma Asp174 and delta Asp180.

Residues gamma Asp174 and delta Asp180, previously implicated in the binding of acetylcholine (ACh) by the mouse muscle ACh receptor, were each mutated to nine other residues, Asn, Glu, Thr, Ala, Cys, His, Val, Tyr, and Lys. The effects of the mutations on ACh-induced current was determined on surface receptors containing wild-type alpha and beta subunits and mutant gamma and delta subunits. The mutations increased the concentration of ACh eliciting half-maximal current (EC50) by factors from 22 for the Glu mutant to 660 for the Lys mutant. Analysis of the effects in terms of the difference in the accessible surface areas of the mutant and wild-type side chains and the difference in side-chain charges indicated that, per binding site, Delta DeltaG0 for activation was a sum of 10 cal mol-1 A-2 of change in side-chain accessible surface area and of 0.95 kcal mol-1 positive step-1 in side-chain charge, equivalent to 1 mol of charge falling through 42 mV. The effects on the concentration of ACh (IC50, ACh) and of d-tubocurarine (IC50,dTC) causing half-maximal retardation of alpha-bungarotoxin binding were determined on complexes containing wild-type alpha and beta subunits and either mutant gamma or mutant delta subunit. The effects on IC50,ACh correlated well with the effects on EC50, with a similar magnitude for the influence of side-chain charge on the free energy of binding (in this case to the desensitized state) and on the electrostatic potential at the binding site. The effects on IC50,dTC were in all cases less than the effects on IC50,ACh, and the two sets of effects were poorly correlated. In line with the higher ACh affinity and lower d-tubocurarine affinity of the alpha-delta binding site compared to the alpha-gamma binding site, mutations of delta Asp180 had a greater effect on IC50,ACh than did the same mutations of gamma Asp174, and vice versa for effects on IC50,dTC. Consequently, all mutations decreased the asymmetry in the binding properties of the two types of sites.

Affinity Labels↗
PubMed1997

Transport bicycles.

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Amino Acid Sequence↗
PubMed1996

The contributions of aspartyl residues in the acetylcholine receptor gamma and delta subunits to the binding of agonists and competitive antagonists.

The acetylcholine (ACh) receptors in muscle have the composition alpha2betagammadelta and contain two ACh binding sites. One is formed between an alpha subunit and the gamma subunit, and the other is formed between an alpha subunit and the delta subunit. Among the residues in the ACh binding sites are alphaCys-192 and alphaCys-193. The negatively charged deltaAsp-180 is at an appropriate distance from alphaCys-192/193 also to be in the ACh binding site and to interact electrostatically with the positively charged ammonium group common to agonists and competitive antagonists. Mutation to Asn of either deltaAsp-180 or the aligned residue in the gamma subunit, gammaAsp-174, decreased the affinities of three agonists, acetylcholine, tetramethylammonium, and succinyldicholine 170-560-fold. By contrast, these mutations decreased the affinities of three competitive antagonists, (+)-tubocurarine, hexamethonium, and dihydro-beta-erythroidine, only 2-15-fold. Agonists, but not antagonists, promote the transitions of the receptor from the resting state to the higher affinity active and desensitized states, and the greater effects of the mutations of gammaAsp-174 and deltaAsp-180 on the apparent affinities of agonists could reflect the involvement of these residues in the conformational changes of the receptor corresponding to its transitions to higher affinity states. In these transitions, one possibility is that gammaAsp-174 and deltaAsp-180 move closer to bound agonist.

Amino Acid Sequence↗
PubMed1996

Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels.

Ligand-gated ion channels are multi-subunit complexes where each subunit-type is encoded by several related genes. Heterologous expression of any one of the neuronal nicotinic acetylcholine receptors (nAChR) alpha-type subunits, either alone or with any beta-type subunit, typically yields functional nAChR channels. A striking exception is the nAChR alpha5 subunit: although apparently complexed with beta2 and beta4 nAChR subunits in neurons, and expressed in a subset of neurons within the central and peripheral nervous systems, heterologous expression of alpha5, either alone or with any beta-type subunit has failed to yield functional channels. We demonstrate here that alpha5 does participate in nAChRs expressed in hetrologous systems and in primary neurons, and further that alpha5 contributes to the lining of functionally unique nAChR channels, but only if coexpressed with both another alpha- and beta-type subunit. Furthermore, channels containing the alpha5 subunit are potently activated and desensitized by nanomolar concentrations of nicotine.

Acetylcholine↗
PubMed1996

Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating.

In voltage-gated ion channels and in the homologous cyclic nucleotide-gated (CNG) channels, the loop between the S5 and S6 transmembrane segments (P region) is thought to form the lining of the pore. To investigate the structure and the role in gating of the P region of the bovine retinal CNG channel, we determined the accessibility of 11 cysteine-substituted P region residues to small, charged sulfhydryl reagents applied to the inside and outside of membrane patches in the open and closed states of the channel. The results suggest that the P region forms a loop that extends toward the central axis of the channel, analogous to the L3 loop of bacterial porin channels. Furthermore, the P region, in addition to forming the ion selectivity filter, functions as the channel gate, the structure of which changes when the channel opens.

Amino Acid Sequence↗
PubMed1996

Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines.

The structure of the NMDA receptor channel M2 segment was investigated by probing the extracellular and cytoplasmic faces of cysteine-substituted NR1-NR2C channels with charged sulfhydryl-specific reagents. The pattern of accessible positions suggests that the M2 segment forms a channel-lining loop originating and ending on the cytoplasmic side of the channel, with the ascending limb in an alpha-helical structure and the descending limb in an extended structure. A functionally critical asparagine (N-site) is positioned at the tip of the loop, and a cluster of hydrophilic residues of the descending limb, adjacent to the tip, forms the narrow constriction of the channel. An apparent asymmetric positioning of the NR1- and NR2-subunit N-site asparagines may account for their unequal role in Ca2+ permeability and Mg2+ block.

Amino Acid Sequence↗
PubMed1995

Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit.

The muscle-type acetylcholine (ACh) receptor has the composition alpha 2 beta gamma delta. The subunits are arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the alpha M2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substituted-cysteine-accessibility method to alpha M1. We analyzed 15 consecutive residues, starting at alpha Pro211 at the extracellular end of M1. Wild-type alpha contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant alpha subunits, together with wild-type beta, gamma, and delta subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: alpha Y213C was susceptible to MSTEA added in the presence or the absence of ACh, alpha P211C, alpha I215C, alpha V216C, alpha N217C, and alpha I220C were susceptible in the absence of ACh, and alpha V218C was susceptible in the presence of ACh. These results imply that M1 is exposed in the channel, and its exposure changes during gating or desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding.

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between alpha-subunit residues Cys192 and Cys193. The cross-linker S-(2-[3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced alpha Cys192/Cys193 and an amide bond with an acidic residue in the delta subunit (Czajkoswski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH2CONHCH2CH2S- is is 0.9 nm long. After the disulfide bond linking -NHCH2CONHCH2CH2S- to the alpha subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the delta subunit by an amide bond. The delta subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to delta Asp165, delta Asp180, and delta Glu182. The mutation of delta Asp180 to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either delta Asp165, delta Glu182, or 8 other acidic residues in the same region of delta decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A. (1993) Proc. Natl. Acad. Sci. U.S.A 90, 6285-6289). Because delta Asp180 both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the alpha and the delta subunits.

Amino Acid Sequence↗
PubMed1995

Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method.

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. We have developed a method to map systematically all the residues forming the surface of this binding-site crevice, and we have applied this method to the third membrane-spanning segment (M3). We mutated, one at a time, 23 residues in and flanking M3 to cysteine and expressed the mutant receptors heterologously. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist. Thus, the side chains of these residues are exposed in the binding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consistent with a short loop followed by six turns of an alpha helix.

Binding Sites↗
PubMed1994

A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

The binding site in G-protein-linked neurotransmitter receptors is formed among their membrane-spanning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membrane-spanning segments which face this water-filled crevice. Human D2 dopamine receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulfhydryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhibited the binding of the D2-specific antagonist [3H]YM-09151-2 to these cells. The highly polar MTS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the receptor, which includes the surface of the binding-site crevice. In contrast, these reagents will have little access to sulfhydryls facing the lipid bilayer or buried in the protein interior. Positively charged MTS reagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of the binding site for positively charged dopamine agonists and antagonists. Furthermore, both agonists and antagonists of the D2 receptor protected against irreversible inhibition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembrane and two extracellular cysteine residues to serine. Only the mutation of Cys118 to serine decreased the susceptibility of antagonist binding to irreversible inhibition by the MTS reagents. Thus, Cys118, a residue in the middle of the third membrane-spanning segment, is exposed in the D2 receptor binding-site crevice.

Amino Acid Sequence↗
PubMed1994

Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates.

All of the potent agonists and competitive antagonists of the acetylcholine receptors are positively charged, onium compounds. Among the interactions involved in the binding of these compounds, electrostatic forces undoubtedly make an important contribution. There is evidence that the acetylcholine binding site contains both acidic and aromatic amino acids. The acidic side chains could provide long-range charge-charge interactions with acetylcholine, while the aromatic side chains could provide short-range cation-pi-electron and hydrophobic interactions. To probe the long-range electrostatic interactions in the binding site, the rate constants for the reactions of sulfhydryl-specific reagents with cysteines in the binding site have been determined as a function of ionic strength. The reagents are the positively charged methanethiosulfonate ethylammonium and methanethiosulfonate ethyltrimethylammonium, the negatively charged methanethiosulfonate ethylsulfonate, and the neutral methyl methanethiosulfonate. In addition, the rate constants of the reactions of these methanethiosulfonates with positively charged, negatively charged, and uncharged simple thiol compounds have been similarly determined. An analysis of these rate constants in terms of absolute rate theory and Debye-Hückel theory is consistent with the acetylcholine binding site containing two to three negative charges and an electrostatic potential at zero ionic strength of about -80 mV relative to bulk solution.

Acetylcholine↗