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Pyrazolopyridazine 1a was identified in a high-throughput screening carried out by BASF Bioresearch Corp. (Worcester, MA) as a potent inhibitor of CDK1/cyclin B and shown to have selectivity for the CDK family. Analogues of the lead compound have been synthesized and their antitumor activities have been tested. A molecular model of the complex between the lead compound and the CDK2 ATP binding site has been built using a combination of conformational search and automated docking techniques. The stability of the resulting complex has been assessed by molecular dynamics simulations and the experimental results obtained for the synthesized analogues have been rationalized on the basis of the proposed binding mode for compound 1a. As a result of the SAR study, monofuryl 1o has been synthesized and is one of the most active compounds against CDK1 of this series.

The present work focuses on the study of the three-dimensional (3D) structural requirements for the leukotriene D4 (LTD4) antagonistic activity of compounds having the basic quinolinyl(bridged)aryl framework. An approach combining pharmacophore mapping, molecule alignment, and CoMFA models was used to derive a hypothesis for a series of LTD4 antagonists having the basic diaryl-bridged framework. In this compound series, the produced pharmacophore hypotheses have shown to yield molecule alignments suitable to derive valuable CoMFA models. Model selection focused on (1) obtention of coherent modeling results, (2) consistency with the available SAR data, and (3) ability to predict the activity of an independent set of congeneric molecules. This approach resulted in a combined pharmacophore and CoMFA model that can generally represent the antagonistic activity within a log unit of the measured value for compounds of the series. The resulting pharmacophore (model C) consists of an acidic or negative ionizable function (AC), a hydrogen-bond acceptor (HBA), and three hydrophobic regions (HY) and produces chemically meaningful alignments with the most active compounds of the series mapping the pharmacophore in a extended energetically favorable conformation.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.

The effect of propionate and methylmalonate on the transport of glycine into rat liver and brain mitochondria was investigated. Both propionate and methylmalonate markedly inhibited mitochondrial glycine transport. These compounds also inhibited14CO2 production from [14C]glycine by isolated brain and liver mitochondria and glycine metabolism in rat brain cortex slices. These results are discussed with reference to hyperglycinaemia associated with propionic acidaemia and methylmalonic acidaemia and as a possible contributory factor to the pathological mechanisms in these conditions.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Cain vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kdof 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Caand that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Cawith moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Kisimilar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Cain smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.

Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2853-2858). To identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on 125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of beta was demonstrated after comparison of reactivities of native beta and single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta, when mutated individually or in combinations, had no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the alpha subunit that allows high affinity ChTX binding.

An inhibitor of apamin binding has been purified to homogeneity in three chromatographic steps from the venom of the scorpion, Leiurus quinquestriatus hebraeus. The inhibitor, which we have named leiurotoxin I, represents less than 0.02% of the venom protein. It is a 3.4-kDa peptide with little structural homology to apamin although it has some homology to other scorpion toxins such as charybdotoxin, noxiustoxin, and neurotoxin P2. Leiurotoxin I completely inhibits 125I-apamin binding to rat brain synaptosomal membranes (Ki = 75 pM). Thus, it is 10-20-fold less potent than apamin. Leiurotoxin I is not a strictly competitive inhibitor of this binding reaction. Like apamin, leiurotoxin I blocks the epinephrine-induced relaxation of guinea pig teniae coli (ED50 = 6.5 nM), while having no effect on the rate or force of contraction in guinea pig atria or rabbit portal vein preparations. Thus, leiurotoxin I of scorpion venom and apamin of honeybee venom demonstrate similar activities in a variety of tissues, yet are structurally unrelated peptides. These two peptides should be useful in elucidating the role of the small conductance, Ca2+-activated K+ channels in different tissues.

BRL 34915 [6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl) 2H-benzo(b) pyran-3-ol], minoxidil sulfate and diazoxide may relax vascular smooth muscle via hyperpolarization due to an opening of membrane potassium channels. We therefore examined the effects of several potassium channel antagonists on the relaxation response to these vasodilators in isolated rat portal venous strips which were mounted in vitro for detecting changes in isometric force. BRL 34915 (IC50 = 4.7 X 10(-8) M), minoxidil sulfate (IC50 = 1.4 X 10(-7) M) and diazoxide (IC50 = 5 X 10(-6) M) elicited concentration-dependent relaxations of the spontaneous, myogenic contractions in venous strips. The relatively nonselective potassium channel antagonists tetraethylammonium ion (0.3-10 X 10(-3) M) and 4-aminopyridine (1-10 X 10(-3) M) caused concentration-dependent shifts (5- to 50-fold) in the relaxation responses to each vasodilator. Charybdotoxin (up to 10(-7) M) and apamin (up to 10(-7) M), known to be antagonists of high and low conductance calcium-activated potassium channels, respectively, had no inhibitory effect on the relaxation-response curves to BRL 34915, minoxidil sulfate or diazoxide. Glyburide (10(-7) to 3 X 10(-5) M), a sulfonylurea which has been shown to block the ATP-modulated potassium channel in insulin-secreting cells, caused concentration-dependent shifts to the right (up to 100-fold) of the IC50 value for BRL 34915 and diazoxide, and at 10(-6) M, abolished the relaxation response to minoxidil sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+ (Kv1) channels in rat cerebellum that are labeled by 125I-margatoxin (125I-MgTX; Kd, 0.08 pM). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1. 1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+ channels in vivo.

Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. Although the size of the beta subunit is that expected for the product of the gene encoding this protein, the size of the alpha subunit is smaller than that predicted from the slo coding region. To determine the basis for this discrepancy, sequence-directed antibodies have been raised against slo. These antibodies specifically precipitate the in vitro translation product of mslo, which yields an alpha subunit of the expected molecular mass (135 kDa). Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH3 cells reveal the presence of an alpha subunit with an apparent molecular mass of 125 kDa. The difference in size of the alpha subunit as expressed in these membranes and the purified preparations is due to a highly reproducible proteolytic decay that occurs mostly at an advanced stage of the maxi-K channel purification. In the purified maxi-K channel preparations investigated, the full-length alpha subunit, an intermediate size product of 90 kDa, and the 65-kDa polypeptide, as well as other smaller fragments can be detected using appropriate antibodies. Proteolysis occurs exclusively at two distinct positions within the long C-terminal tail of slo. In addition, evidence for the tissue expression of distinct splice variants in membrane-bound as well as purified maxi-K channels is presented.

The interactions between flurbiprofen (FB) and different polymers are studied in order to improve the bioavailability of FB. FB–polymer phase diagrams [poly(ethylene glycol) (PEG) 4000, PEG 6000, and poly-ε-caprolactone] were constructed and compared with the modeling diagrams. Thermoanalytical methods (differential scanning calorimetry, thermomicroscopy) were used to construct the phase diagrams. Thermodynamic data were used to model the FB–polymer systems. The construction of the FB–polymer phase diagrams showed the existence of a stable invariant called “eutectic” characterized by (XE)exp, (TfE)exp, and (ΔHfE)exp, the experimental eutectic composition, the experimental temperature, and the enthalpy of eutectic melting, respectively. Modeling confirmed the values for these parameters and was used to evaluate the different Flory–Huggins parameters χ for each FB–polymer mixture. χ values and the infrared spectra confirm that the interactions due to hydrogen bonds between FB and PEG 4000 are more numerous than between FB and PEG 6000 and also more numerous than between FB and poly-ε-caprolactone.

When purified porcine cardiac sarcolemmal membrane vesicles are incorporated into planar lipid bilayers formed at the tip of patch electrode pipettes, individual divalent cation channels can be monitored. Channel activity is increased in the presence of the Ca2+ channel agonist Bay K 8644, is voltage dependent, and selects for divalent cations over anions. The activity does not inactivate because it is maintained during prolonged depolarizations. Determination of divalent cation selectivity from the reversal potential of single-channel currents indicates a relative permeability ratio for Ba/Ca/Mg of 1:0.45:0.08. Mean channel conductance in 0.1 M Ba2+/0.01 M Mg2+ is 8 pS. Channels are reversibly blocked by the Ca2+ channel inhibitor nitrendipine, and inhibition can be competitively antagonized by Bay K 8644. Binding studies with 3H-labeled D-600 demonstrate the presence of high-affinity receptors for D-600 in sarcolemmal membranes (Kd = 6.4 X 10(-9) M; Bmax = 3 pmol per mg of protein). In addition, experiments with resolved D-600 stereoisomers indicate that (-)D-600 is at least 25-fold more potent than (+)D-600 in competing for this aralkyl amine receptor. Consistent with this, (-)D-600 is much more effective than the (+) isomer in inhibiting bilayer-incorporated channels. These results demonstrate that the divalent cation channel that has been reconstituted in planar lipid bilayers possesses many of the characteristics of voltage-regulated Ca2+ channels in heart and suggest that receptors for Ca2+ entry blockers are functionally associated with this channel.

Airway smooth muscle plasma membranes are rich in K+ channels of various types. Charybdotoxin (ChTX) is a potent blocker of the high-conductance Ca(++)-activated K+ channel in smooth muscle and produces a concentration-dependent contraction of guinea pig trachea. In the present study, pharmacologic experiments were performed on carbachol-contracted (0.34 microM) guinea-pig trachea contracted further with ChTX in order to determine if Ca(++)-activated K+ channels play a role in the responses to cAMP-dependent and cAMP-independent bronchodilators. Relaxation concentration response curves to the beta-agonists, isoproterenol and salbutamol; the phosphodiesterase inhibitor, aminophylline; the cAMP mimic, N6-2'-O-adenosine 3':5'-cyclic monophosphate the guanylate cyclase activator, sodium nitroprusside; and the K+ channel agonists, BRL-34915 and pinacidil, were obtained in the absence and presence of ChTX. The concentration response curves to isoproterenol and salbutamol were shifted to the right (approximately 27-fold and greater than 40-fold, respectively) by 180 nM ChTX, whereas concentration response curves to N6-2'-O-adenosine 3':5'-cyclic monophosphate and aminophylline were affected significantly less (shifted approximately 7.5-fold). Concentration response curves to the cGMP-dependent relaxant sodium nitroprusside were also altered by ChTX (17-fold rightward shift at 180 nM). In the presence of 60 nM ChTX, the concentration response curves to the above relaxants were shifted only 3- to 5-fold. In contrast, ChTX (60 and 180 nM) failed to produce a significant rightward shift in the concentration response curves to BRL-34915 or pinacidil. Relaxation to BRL-34915 was however, blocked by glybenclamide, suggesting differences in the mechanism of relaxation. Contraction of tissues with depolarizing concentrations of KCl (20-80 mM) inhibited responses to all bronchodilators. These results suggest that hyperpolarization of tracheal smooth muscle as a result of opening various types of K+ channels can lead to relaxation of carbachol-contracted tracheal smooth muscle.

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kdof 0.6 nMand a Bmaxthat is in approximately 1:1 stoichiometry with other Ca2+entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+channel effectors. Metal ion channel inhibitors (e.g.Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+entry blockers partially inhibit binding with Kivalues equivalent to their respective Kdvalues, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kdand Bmaxof fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g.pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Kivalues much reduced from their measured Kdvalues, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+flux through L-type Ca2+channels in GH3cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+channel inhibitors.

Five novel peptidyl inhibitors of Shaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus. The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli. HgTX1 inhibits 125I-margatoxin binding to rat brain membranes as well as depolarization-induced 86Rb+ flux through homotetrameric Kv1.1, Kv1. 2, and Kv1.3 channels stably transfected in HEK-293 cells, but it displays much lower affinity for Kv1.6 channels. A HgTX1 double mutant (HgTX1-A19Y/Y37F) was constructed to allow high specific activity iodination of the peptide. HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting 125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values approximately 0.3 pM). 125I-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels and to rat brain membranes (Kd values 0.1-0.25 pM, respectively) but with lower affinity to Kv1.6 channels (Kd 9.6 pM), and it does not interact with either Kv1.4 or Kv1.5 channels. Several subpopulations of native Kv1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using antibodies specific for Kv1 subunits. HgTX1 represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and Kv1 subunit assembly in different tissues.

Tetrandrine, a bis-benzylisoquinoline alkaloid derived from the Chinese medicinal herb Stephania tetrandra, is a putative Ca2+ entry blocker whose mechanism of action is unknown. To investigate this mechanism, the effects of tetrandrine were characterized on binding of three chemical classes of Ca2+ entry blockers in cardiac sarcolemmal membrane vesicles. In the range 25-37 degrees C, tetrandrine completely blocks diltiazem binding, partially inhibits D-600 binding, and markedly stimulates nitrendipine binding, with greatest enhancement occurring at 37 degrees C. The potency of tetrandrine is increased 10-fold as temperature is raised from 25 to 37 degrees C. Scatchard analyses indicate that inhibition of diltiazem binding and stimulation of nitrendipine binding result from changes in ligand affinities while inhibition of D-600 binding is due to both an increase in KD and decrease in Bmax of aralkylamine receptors. Ligand dissociation studies reveal that tetrandrine increases D-600 off-rates, decreases nitrendipine off-rates, but has no effect on diltiazem dissociation kinetics. In addition, tetrandrine reversibly blocks inward Ca2+ currents through L-type Ca2+ channels in GH3 anterior pituitary cells. These results indicate that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+ entry blocker receptor complex and allosterically modulates ligand binding at other receptors in this complex. These findings suggest that tetrandrine is a structurally unique natural product Ca2+ entry blocker and provide a rationale explanation for the therapeutic effectiveness of this agent.

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.