Diffusion model in ion channel gating. Extension to agonist-activated ion channels.

Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165-1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude.     

Activation of a nicotinic acetylcholine receptor.

We studied activation of the nicotinic acetylcholine (ACh) receptor on cells of a mouse clonal muscle cell line (BC3H1). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (beta) and agonist dissociation (k-2). We find that this estimate of beta does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 microM; Carb: 300-1,000 microM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state.     

Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells.

Parameters associated with activation and desensitization of the nicotinic receptor in the BC3H-1 muscle cell line have been compared with the state transitions that result upon combination with agonist. 125I-labeled cobra alpha-toxin is found to bind to an apparent single class of surface nicotinic receptors on the cells in situ with a rate constant of 1.15 x 10(5) M-1 s-1. The competition between cholinergic ligands and alpha-toxin reveals that agonists, but not classical antagonists, will promote a slow conversion to a receptor state where the affinity for agonists is enhanced. Moreover, agonists such as carbamylcholine elicit a permeability increase to 22Na+ ions that slowly decrements at a rate and to an extent closely paralleled by the conversion of the receptor to the high affinity state. Upon removal of the agonist, both the affinity increase and the diminished permeability change are completely reversible and again exhibit similar kinetics for their return to the original state. A comparison of the capacity of full agonists to compete with alpha-toxin binding and elicit a permeability change suggests that in the absence of agonist, receptor predominates in a low affinity activatable state. Binding of agonists to the low affinity state exhibits little if any cooperativity (n = 0.97 to 1.31), while the corresponding permeability change appears more cooperative (n = 1.31 to 1.52). By contrast, when receptors have been previously equilibrated with agonists, occupation of the receptor occurs over a 3- to 5-fold lower concentration range. Binding following equilibration closely correlates with a concomitant decrease in activatable receptor resulting from equivalent exposure to agonist. Furthermore, under equilibrium conditions, the binding of full agonists is typified by a moderate degree of homotropic cooperativity (1.25 to 1.44), enabling the receptor to desensitize over a narrow range of agonist concentration. Simultaneous measurement of occupation and activation parameters has enabled us to compare a state function for desensitization which is generated from binding parameters with the reduction in permeability seen in the desensitization process. A scheme describing the association of agonist with two functionally distinct receptor states is developed to account for the cooperative relationship between agonist binding and desensitization of the receptor.     

Direct measurement of the concentration- and time-dependent open probability of the nicotinic acetylcholine receptor channel.

Using the outside-out patch clamp recording technique together with a rapid solution exchange system, we measured ionic currents through nicotinic acetylcholine (ACh) receptor channels from BC3H-1 cells in response to rapid applications of 0.3-1,000 microM ACh. We used nonstationary fluctuation analysis of ensembles of responses to deduce the number of channels in the patch, the maximum open channel probability as a function of ACh concentration and the time course of a fast desensitization process. We found that: (a) Excised patches from BC3H-1 cells typically contain between 50 and 150 functional ACh receptor ion channels. (b) The open channel probability is proportional to [ACh]1.95 at low concentrations of ACh, is half-maximal at 20 microM ACh and saturates above 100 microM ACh. (c) ACh is a very efficacious agonist; 100 microM ACh opens at least 90% of the available channels. This estimate of efficacy is model-independent. (d) The rate of decay of the agonist-induced current is concentration-dependent. In the presence of 100 microM ACh the current decays with a time constant of 50-100 ms. It decays more slowly in the presence of lower concentrations of agonist but is relatively insensitive to voltage.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

The role of loop 5 in acetylcholine receptor channel gating

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse alpha-subunit 92-100) that links the beta4 and beta5 strands of the extracellular domain and that (in the alpha-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to alphaD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most alphaD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that alphaD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Phi = 0.93, which implies an open-like character at the transition state). alphaD97 mutations in the two alpha-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of alphaD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ε-subunit DNA

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of -,β-, and δ-subunits [1]. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ε-subunit, which is believed to replace the γ-subunit in forming the adult AChR subtype [4], BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR ε-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ε-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant ε-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ε-subunit may assemble with mouse muscle -, β- and δ-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ε-subunit mRNA. Thus, the notion that replacement of the γ-subunit by the ε-subunit during development is the result of the postnatal rise in the level of ε-subunit specific mRNA is further supported.     

The temperature dependence of some kinetic and conductance properties of acetylcholine receptor channels

We examined the temperature dependence of single-channel properties of the nicotinic acetylcholine receptor channel from clonal BC3H-1 cells over a range of 10-40 degrees C. We found temperature sensitivities (Q10 values) of 2-4 for the mean channel open time. The Q10 did not depend strongly on voltage and the voltage dependence of the mean open time was temperature-independent. The Q10 of closing rate of the long-lived open state was 3-4 but the Q10 of closing rate of the brief open state was independent of temperature. The duration of brief closures could be measured only between 10 and 25 degrees C. Since this approached the limit of the experimental time resolution, an accurate determination of the Q10 could not be made. The current decay due to desensitization after rapid application of high concentrations of agonist varied with a Q10 of about 2. The conductance of single channels (the inverse of the ion translocation rate) had a Q10 of 1.3-1.5. We found no obvious nonlinearities in the Arrhenius curves for any of the measured properties.     

Myogenic programs of mouse muscle cell lines: expression of myosin heavy chain isoforms, MyoD1, and myogenin

Different mouse muscle cell lines were found to express distinct patterns of myosin heavy chain (MHC) isoforms, MyoD1, and myogenin, but there appeared to be no correlation between the pattern of MHC expression and the patterns of MyoD1 and myogenin expression. Myogenic cell lines were generated from unconverted C3H10T1/2 cells by 5-azacytidine treatment (Aza cell lines) and by stable transfection with MyoD1 (TD cell lines) or myogenin (TG cell lines). Myogenic differentiation of the newly generated cell lines was compared to that of the C2C12 and BC3H-1 cell lines. Immunoblot analysis showed that differentiated cells of each line expressed the embryonic and slow skeletal/beta-cardiac MHC isoforms though slow MHC was expressed at a much lower, barely detectable level in BC3H-1 cells. Differentiated cells of each line except BC3H-1 also expressed an additional MHC(s) that was probably the perinatal MHC isoform. Myogenin mRNA was expressed by every cell line, and, with the exception of BC3H-1 (cf., Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Cell. 51:987-1000), MyoD1 mRNA was expressed by every cell line. To determine if MyoD1 expression would alter the differentiation of BC3H-1 cells, cell lines (termed BD) were generated by transfecting BC3H-1 cells with MyoD1 under control of the beta-actin promoter. The MyoD1 protein expressed in BD cells was correctly localized in the nucleus, and, unlike the parental BC3H-1 cell line that formed differentiated MHC-expressing cells, which were predominantly mononucleated, BD cell lines formed long, multinucleated myotubes (cf., Brennan, T. J., D. G. Edmondson, and E. N. Olson. 1990. J. Cell. Biol. 110:929-938). Despite the differences in morphology and MyoD1 expression, BD myotubes and the parent BC3H-1 cells expressed the same pattern of sarcomeric MHCs.     

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B4 and inhibited by inhibitors of arachidonic acid oxidation

The mechanism of agonist-induced desensitization of the beta adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both alpha and beta adrenergic receptors and nicotinic receptors. beta receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37 degrees but not at 4 degrees, induced a dose dependent internalization of the beta adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. beta adrenergic receptor internalization was very specific: the alpha adrenergic agonist Phenylefrine had almost no effect on beta receptor levels, while Isoproterenol treatment had no effect on the number of alpha adrenergic or nicotinic receptors expressed at the cell surface of these cells. beta adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.     

Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating.

Affinity labeling and mutagenesis studies have demonstrated that the conserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subunit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutations at Y190. In Y190F the dissociation constant for ACh binding to closed channels was reduced approximately 35-fold at the first binding site and only approximately 2-fold at the second site. At both binding sites the association and dissociation rate constants were decreased by the mutation. Compared with wildtype AChRs, doubly-liganded alpha Y190F receptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully occupied-open), there is an increase of approximately 6.4 kcal/mol caused by the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes from altered agonist binding to the first and second binding sites, respectively. The closing rate constant of alpha Y190F receptors was the same with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was approximately 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel block by ACh was approximately 2-fold lower in alpha Y190F receptors compared with in wildtype receptors, suggesting that there are changes in the pore region of the receptor as a consequence of the mutation. The activation reaction is discussed with regard to energy provided by agonist-receptor binding contacts, and by the intrinsic folding energy of the receptor.     

Analysis of cyclic and acyclic nicotinic cholinergic agonists using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy.

The relationship between the structure and function of a series of nicotinic cholinergic agonists has been studied using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. The cyclic compound 1,1-dimethyl-4-acetylpiperazinium iodide and its trifluoromethyl analogue (F3-PIP) interact with nicotinic acetylcholine receptors (nAChRs) from both Torpedo electroplaque and BC3H-1 cells at lower concentrations than the acyclic derivatives, N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide and its fluorinated analogue (F3-TED). The magnitude of the difference in potencies depends on the type of measurement. In binding experiments, the differences between the two classes of compounds depends mainly on the conditions of the experiment. In measurements of the initial interaction with the nAChR, the PIP compounds have an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incubations indicated that the PIP compounds were able to induce a time-dependent shift in receptor affinity consistent with desensitization, whereas the TED compounds were unable to induce such a shift. The activation of single channel currents by the cyclic compounds occurs at concentrations approximately two orders of magnitude lower than for the acyclic compounds, but the TED compounds exhibit a larger degree of channel blockade than the PIP compounds. Previous work (McGroddy, K.A., and R.E. Oswald. 1992. Biophys. J. 64:314-324) has shown that the TED compounds can exist in two energetically distinct conformational states related by an isomerization of the amide bond. 19F nuclear magnetic resonance experiments suggest that the higher energy population of the TED compounds may interact preferentially with the ACh binding sites on the nAChRs and that a significant fraction of the difference between the initial affinity of the PIP and TED compounds may be accounted for by the predominance in solution of a conformational state less able to interact with the ACh binding sites on nAChRs.     

alpha 7-type acetylcholine receptor localization and its modulation by nicotine and cholesterol in vascular endothelial cells

The neuronal‐type α7 nicotinic acetylcholine receptor (α7AChR) is also found in various non‐neural tissues, including vascular endothelium, where its peculiar ionotropic properties (high Ca²⁺ permeability) and its supervening Ca²⁺‐mediated intracellular cascades may play important roles in physiology (angiogenesis) and pathology (inflammation and atherogenesis). Changes in molecular (up‐regulation, affinity, and conformational states) and cellular (distribution, association with membranes) properties of the α7AChR related to angiogenesis (wound‐repair cell migration) and atherogenesis (alterations in cholesterol content) were studied in living endothelial cells, with the aim of determining whether such changes constitute early markers of inflammatory response. The combination of pharmacological, biochemical, and fluorescence microscopy tools showed that α7AChRs in rat arterial endothelial (RAEC) and human venous endothelial (HUVEC) cells occur at extremely low expression levels (～50 fmol/mg protein) but undergo agonist‐induced up‐regulation at relatively high nicotine concentrations (～300‐fold with 50 µM ligand), increasing their cell‐surface exposure. When analyzed in terms of cold Triton X‐100 solubility and subcellular distribution, α7AChRs occur in the “non‐raft” subcellular membrane fractions. Acute cholesterol depletion reduced not only cholesterol levels but also the number of cell‐surface α7AChRs. Nicotine exposure markedly stimulated cell migration and accelerated wound repair, which drastically diminished in cells deprived of the sterol. The angiogenic effect of nicotine appears to be synergistic with cholesterol content. Finally, the apparent K_D of α7AChRs for the open‐channel blocker crystal violet was found to be ～600‐fold lower in receptor‐enriched membranes obtained from up‐regulated HUVEC. J. Cell. Biochem. 112: 3276–3288, 2011. © 2011 Wiley Periodicals, Inc.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

Enhancement of DNA synthesis in rat thymocytes by stimulating their muscarinic acetylcholine receptors.

The binding of 3H-acetylcholine (ACh) to acetylcholine receptors (AChRs) on rat thymocytes was examined and found to be inhibited by the treatment with several antagonists against nicotinic and muscarinic AChRs. This result suggested that thymocytes have AChRs with different affinity, and bear both nicotinic and muscarinic AChRs on their surfaces. To make clear the functional significance of the AChRs, DNA synthesis of the thymocytes stimulated with ACh was examined. 3H-thymidine uptake of thymocytes was significantly increased when the cells were stimulated with ACh or another cholinergic agonist. The increment of DNA synthesis caused by ACh in thymocytes was not reduced by treatment with nicotinic antagonists, but was decreased by treatment with any of the muscarinic antagonists. Concentration of the intracellular second messengers, inositol 1,4,5-triphosphate (IP3) and guanosine 3',5'-cyclic monophosphate (cGMP) was also made higher by ACh stimulation. It is discussed that the enhancement of intracellular IP3 and cGMP concentrations after stimulation of muscarinic AChRs appears to be related with the increment of thymocyte DNA synthesis.     

Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.