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K W Miller

Publications and source records attributed to K W Miller.

At least 55 records · Page 3Linked to original sources
PubMed1995

Channel inhibition by alkanols occurs at a binding site on the nicotinic acetylcholine receptor.

The mechanism by which normal alkanols longer than ethanol inhibit cation flux through the transient open state of the nicotinic acetylcholine receptor (nAcChoR) is unknown. They might act nonspecifically either by perturbing the lipid bilayer or by binding to many low affinity sites. Alternatively, they might act in a mutually exclusive manner at a well defined site on the protein. To address this problem, a rapid assay of agonist-induced 86Rb+ efflux from nAcChoR-rich Torpedo membrane vesicles was used that enabled the anesthetic-induced inhibition to be measured on a millisecond time scale, under conditions where the concentration of all ligands was raised in < 1 msec, thereby avoiding complications due to desensitization. By measuring the inhibition constant of one agent as a function of the fixed concentration of a second agent, it is possible to distinguish between nonspecific action and mutually exclusive action. Our data are inconsistent with the hypothesis that 1-octanol and 1-heptanol act in a nonspecific manner, but they are consistent with the hypothesis that these two alkanols act in a mutually exclusive manner at a well defined site. The data suggest that the alkanols sterically compete for the site, but experimental limitations prevented a less plausible model, in which there is a strong negative allosteric interaction between separate octanol and heptanol sites, from being ruled out. Should the latter interaction occur, the data indicate that occupation of one alkanol site would decrease the affinity of the other by about 50-fold. The local anesthetic procaine is known to act in a mutually exclusive manner with the agonist self-inhibition site. We found that octanol and procaine acted as separate sites, which exhibited a negative heterotrophic interaction such that octanol reduced the affinity of procaine 6-fold. We conclude that octanol and heptanol inhibit cation flux through the channel of the nAcChoR by binding to a site (or a set of sites of equal affinity) whose location is distinct from, but allosterically coupled to, the agonist self-inhibition site.

1-Octanol↗
PubMed1995

Molecular sites of general anaesthetic action on acetylcholine receptors.

Mechanisms of anaesthetics still remain unclear. However, various attempts have been made to elucidate the effects of anaesthetics at the molecular level. The nicotinic acetylcholine receptor has proved to be a good model of membrane-bound ligand-gated ion channels. It is available in abundance from Torpedo electroplaques, thus enabling multiple experimental approaches. The receptor exists in different states: the resting state, the open state and the desensitized state, amongst others. For each of these states, effects of general anaesthetics at the receptor molecule have been shown. Displacement studies show barbiturates to bind to a site on the resting state of the nicotinic acetylcholine receptor; cation flux studies suggest barbiturates may also bind to the same, or a similar, site on the open state of the receptor when inhibiting its function. Long-chain alcohols inhibit the open receptor, perhaps by binding to such a site or sites. However, short-chain alcohols do not inhibit and do not share this long-chain alcohol binding site; instead they nonspecifically enhance the agonist's apparent affinity. All the alcohols also cause desensitization by a non-specific mechanism possibly involving perturbation of the lipid bilayer. Thus, general anaesthetics exert both specific and non-specific actions on the acetylcholine receptor.

Alcohols↗
PubMed1994

A hydrophobic inhibitor of the nicotinic acetylcholine receptor acts on the resting state.

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Lipid-protein interactions and protein dynamics in vesicles containing the nicotinic acetylcholine receptor: a study with ethanol.

Electron paramagnetic resonance (EPR) spectroscopy was used to study the action of ethanol on the protein side chain motions of the nicotinic acetylcholine receptor (nAcChoR) in alkaline extracted membranes from Torpedo nobiliana. EPR spectra of the nAcChoR derivatized with maleimide spin label contain both strongly and weakly immobilized components. The rotational correlation time of the strongly immobilized component decreases by a factor of 2-3-fold with the addition of 1.6 M ethanol, while that of the weakly immobilized component is not significantly altered. EPR spectroscopy was also used to probe the lipid environment immediately surrounding the nAcChoR with stearic acid and phosphatidylcholine spin labeled at the fourteenth acyl carbons (14-SASL and 14-PCSL, respectively), and the steroid spin label androstanol (ASL). EPR spectra of these probes reveal a component corresponding to lipids that are motionally restricted by the receptor (annular lipids) in addition to a more fluid component arising from bulk lipid. Using spectral subtraction, the order of selectivity of these spin labels for the nAcChoR was determined to be ASL > or = 14-SASL > 14-PCSL. The estimated rotational correlation times of the high affinity 14-SASL and ASL probes ranged from approx. 20 to 35 ns. The correlation times of the lower affinity 14-PCSL were generally shorter than those for 14-SASL and ASL and ranged from about 10 to 25 ns. The addition of up to 0.9 M ethanol altered neither the affinity nor the mobility of the motionally restricted EPR component. This suggests that ethanol's actions on the nAcChoR are not mediated via changes at the lipid/protein interface near the center of the bilayer.

Animals↗
PubMed1994

Ethanol enhances agonist-induced fast desensitization in nicotinic acetylcholine receptors.

The reversible decline of the nicotinic acetylcholine receptor's response to acetylcholine during prolonged exposure to acetylcholine is known as desensitization. Here, we studied ethanol's modulation of fast agonist-induced desensitization of the nicotinic acetylcholine receptor in postsynaptic membrane vesicles from Torpedo using a fast kinetic technique: pulsed quenched flow. Preincubation of the vesicles with various concentrations of acetylcholine at 4 degrees C for times ranging from 80 ms to 1.5 s caused fast desensitization, which was revealed as a decreased 86Rb+ influx when the vesicles were subsequently briefly exposed to a saturating concentration of acetylcholine in 86RbCl. Acetylcholine-induced fast desensitization had a maximum observed rate, kdmax, of 6.8 s-1, a half-effect concentration, KD, of 157 microM, and a Hill coefficient of 1.4. Increasing the ethanol concentration up to 1.0 M causes a linear increase in kdmax, such that 1.0 M ethanol doubles the rate. Ethanol (1 M) also decreased KD 10-fold without changing the Hill coefficient. We consider a modified sequential model to interpret our data. Two acetylcholine molecules bind sequentially to the receptor's resting state to form a pre-open (closed) state, which then opens and, at very high acetylcholine concentrations, is inhibited. A priori fast desensitization might occur from any of these acetylcholine-occupied states. If we assume fast desensitization to occur solely from the pre-open state, our data predict an excessively large action of ethanol on the fast desensitization rate constant (> 200-fold increase in the desensitization rate constant at 1 M ethanol). When we assume fast desensitization to occur from all states, ethanol is seen to have two actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Does general anesthetic-induced desensitization of the Torpedo acetylcholine receptor correlate with lipid disordering?

We have tested the hypothesis that general anesthetics stabilize the desensitized state of the nicotinic acetylcholine receptor by disordering its surrounding lipids. Acetylcholine receptor-rich postsynaptic membranes from the electroplaques of Torpedo were used in this study to obtain the highest possible receptor specific activity in native membranes. We examined 18 general anesthetics, including six inhalation agents, eight 1-alcohols, the enantiomers of 2-octanol, and two intravenous general anesthetics (pentobarbital and ethylcarbamate). The degree of desensitization after preincubation with the general anesthetics was determined by brief exposure to [3H]acetylcholine, making use of the facts that desensitized receptors have much higher affinity than do those in the resting state and that interconversion between the states is slow. All of the general anesthetics desensitized the receptor within minutes, exhibiting steep concentration-response curves with Hill coefficients generally within the range of 2-4. At the highest general anesthetic concentrations, almost all receptors were desensitized. The concentrations that desensitized half of the resting state receptors varied by > 3000-fold. The 2-octanol enantiomers were without stereoselectivity. Membrane order was examined in parallel by using spin-labeled fatty acids doped into the native membranes. The spin label 5-doxylpalmitate reported from the most ordered part of the bilayer near the aqueous interface, whereas 12-doxylstearate reported from the less ordered region nearer the center of the bilayer. The spin label deeper in the membranes was 3 times more sensitive to a given anesthetic than was the other probe. At both depths in the membrane general anesthetics decreased lipid order linearly with increasing concentration. The range of disordering potencies (change in order parameter induced by a unit concentration of general anesthetic in the aqueous phase) was 5333 for 5-doxylpalmitate and 7143 for 12-doxylstearate, but the range of disordering compared at equally desensitizing concentrations was reduced by 875- and 1430-fold, respectively. The average degrees of disordering at concentrations that desensitized half of the resting state receptors were 1.5% and 4.4%, respectively. It is unlikely that changes in membrane order parameter per se cause desensitization, because the associated changes in order parameter can be reproduced by changes in cholesterol content or temperature that do not cause desensitization. We conclude that, although there is a strong association between anesthetic-induced membrane disordering and desensitization, more detailed tests of a mechanistic nature will be necessary to elucidate the mechanisms underlying the Meyer-Overton-type behavior we have observed.

Anesthesia, General↗
PubMed1994

Ethanol stabilizes the open channel state of the Torpedo nicotinic acetylcholine receptor.

Ethanol is known to cause a leftward shift of the acetylcholine concentration-response curve for channel opening of the nicotinic acetylcholine receptor (nAcChoR). However, it remains uncertain whether the mechanism underlying ethanol's effect is an increase in the binding affinity of the agonist to the receptor or an increase in the open/closed equilibrium for those receptors occupied by agonist. In the present study, this question was resolved by measuring the efflux of 86Rb+ over 9 msec from Torpedo vesicles after rapid mixing with the partial agonist suberyldicholine with or without ethanol as appropriate. Suberyldicholine's concentration-response curve is bell-shaped. Two actions underlie this bell-shaped curve, namely activation at low concentration (apparent dissociation constant for activation, Ka = 38 microM) and self-inhibition at higher concentration (apparent dissociation constant for inhibition, Kb = 9 mM), but the overlap of these two actions only reduces the maximum observable flux by 20%. Increasing ethanol concentration from 0 to 0.9 M causes: a linear increase in the maximum response of the nAcChoR to suberyldicholine from 5 to 80% of the maximum induced by acetylcholine, a moderate increase in Ka, and no change in Kb. Analysis of our results using the sequential two-site binding model revealed that the main action of ethanol on nAcChoR was to increase the fraction of occupied receptors that open. The equilibrium constant describing this effect changed by 8-fold at anesthetic concentrations. Ethanol also decreased the affinity of suberyldicholine for its self-inhibition site by a comparable amount, suggesting that its main action is to stabilize the open state. In addition, ethanol caused a small increase in suberyldicholine's affinity for the agonist site.

Animals↗
PubMed1993

Membrane protein topology determination by proteolysis of maltose binding protein fusions.

A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest. Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain. A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm. A cytosolic MBP domain (MBP delta 2-26) is attached to the protein when its N-terminus resides in the cytoplasm. The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop. Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP. The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments. Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain. The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments. Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them.

ATP-Binding Cassette Transporters↗
PubMed1993

Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: evidence for specific protein structural changes upon desensitization.

We have previously reported a new method based on Fourier transform infrared spectroscopy for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR) [Baenziger, J. E., Miller, K. W. & Rothschild, K. J. (1992) Biophys. J. 61, 983-992; Baenziger, J. E., Miller, K. W., McCarthy, M. P. & Rothschild, K. J. (1992) Biophys. J. 62, 64-66]. Spectra are recorded using attenuated total reflection both in the presence and absence of agonists. The resulting nAChR "resting-to-desensitized" difference spectra reveal small highly reproducible infrared bands which can arise from vibrations of the agonist and structural changes in the nAChR membrane during the conversion of the receptor from the resting to desensitized state. In this work we have used a combination of different agonists and an antagonist along with isotopic labeling to assign bands in these spectra. nAChR membranes pretreated with the competitive antagonist alpha-bungarotoxin exhibit no bands above the noise level (approximately 10(-5) au) demonstrating that vibrations of the unbound agonist do not contribute to the normal difference spectrum. In contrast, bands in the resting-to-desensitized difference spectra are identified which can be assigned to the bound agonist, providing a means to probe its interaction and orientation in the binding site. Additional difference bands are due to secondary structural changes of the protein, perturbation of tyrosine(s), and changes in carboxyl groups possibly from Asp and/or Glu residues. Remarkably, some of these spectral changes are similar to those detected during the bleaching of the photoreceptor membrane protein rhodopsin.

Animals↗
PubMed1993

The effect of general anesthetics on the dynamics of phosphatidylcholine-acetylcholine receptor interactions in reconstituted vesicles.

The interaction of general anesthetics at the lipid/protein interface of the nicotinic acetylcholine receptor reconstituted in dioleoylphosphatidylcholine bilayers at various lipid/protein ratios has been studied using the electron spin resonance spectra of phosphatidylcholine spin-labeled at the fourteenth acyl carbon (14-PCSL). In addition to the bilayer spectrum, the spin label reported a more motionally restricted environment whose contribution increased with increasing protein/lipid ratio. Exchange between these two environments occurred at a rate of approx. 6 x 10(7) s-1. The motionally restricted, protein-associated 14-PCSL had a rotational correlation time of about 10-20 ns, an order of magnitude slower than when in the bilayer. Addition of 1-hexanol (up to 16 mM) to the reconstituted receptor perturbed the acyl chains of the bulk lipid phase, but the motional properties of the lipid acyl chains at the protein/lipid interface near the membrane center were not significantly perturbed on the EPR motional time-scale. Similarly, anesthetics that were less effective at perturbing the bilayer, such as pentobarbital (up to 2 mM) and isoflurane (7 mM), did not perturb the lipid/protein interface on the conventional EPR motional time scale.

Anesthesia, General↗
PubMed1993

Trans-unsaturated lipid dynamics: modulation of dielaidoylphosphatidylcholine acyl chain motion by ethanol.

Acyl chain dynamics of the trans-unsaturated lipid, dielaidoylphosphatidylcholine (DEPC), were studied by conventional and saturation transfer electron paramagnetic resonance spectroscopy of aqueous dispersions of DEPC spin labeled with lecithins having doxyl groups at positions 5, 10, and 14 on the sn-2 chain. The gel to liquid crystalline transition is concerted with simultaneous increases in rotational motion about the long axis of the acyl chain (libration) and in gauche-trans conformational interconversions (wobble). Relative to saturated lecithins at similar reduced temperatures the double bond (a) slowed libration by an order of magnitude in both phases, while wobble motions were several times slower, and (b)-produced a pronounced stiffness of the acyl chain near the double bond. Ethanol (0-1.6 M), in addition to its well-known colligative effect on the phase transition, was found to decrease the bilayer order in a concentration-dependent manner. This effect was smaller in the gel than in the liquid crystalline phase, most pronounced next to the double bond, and weakest deep in the bilayer. Ethanol affected slow motions little in the gel phase but wobble and libration correlation times were markedly decreased in the liquid crystalline phase.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

The role of charge in lipid selectivity for the nicotinic acetylcholine receptor.

The effect of salt and pH titration on the selectivity of spin-labeled analogues of phosphatidic acid, phosphatidylserine, phosphatidylcholine, and stearic acid for the nicotinic acetylcholine receptor (nAcChoR) reconstituted into dioleoylphosphatidylcholine was examined at 0 degrees C using electron spin resonance spectroscopy. The order of selectivity at pH 7.4 and 0 mM NaCl was phosphatidylserine > stearic acid > phosphatidic acid > phosphatidylcholine. The addition up to 2 M NaCl or titration of pH from 5.0 to > 9.0 did not alter the selectivity of the phospholipids for the nAcChoR. For stearic acid, conversely, titration of pH from 5.0 to 9.0 at 0 mM NaCl and titration of NaCl from 0 to 2 M at pH 9.0 both increased selectivity for the nAcChoR. It is concluded that electrostatic interactions do not account for the selectivity of the negatively charged phospholipids, phosphatidylserine, and phosphatidic acid for the nAcChoR. This is consistent with the known orientation of the transmembrane sequences M1 and M4, which predicts a balance in the number of negative and positive charges in the lipid-protein interface and suggests that the two positive charges on each M3 helix are not exposed to the lipid-protein interface.

Animals↗
PubMed1993

Conjunctival impression cytology as an indicator of vitamin A status in lactating Indonesian women.

Conjunctival impression cytology (CIC) has been used to assess the vitamin A status of children but not women. We used CIC in a randomized controlled trial of high-dose vitamin A supplementation in Indonesian women. We report its association with biochemical indicators and evaluate its usefulness for several assessment functions, using two definitions for abnormal CIC. Serum retinol concentrations of all women and milk vitamin A concentrations of unsupplemented women decreased with worsening CIC category. CIC indicated risk of low milk vitamin A and low infant serum retinol with low sensitivity and high specificity. CIC did not identify women who benefited from supplementation. The usefulness of CIC to measure response to supplementation was limited by the low prevalence of abnormal status in these women. CIC may be a useful indicator of vitamin A deficiency in women or their breast-fed infants, or to measure response to supplementation, when a more sensitive cutoff point for abnormal status is used.

Conjunctiva↗
PubMed1993

High dose vitamin A supplementation of breast-feeding Indonesian mothers: effects on the vitamin A status of mother and infant.

For regions where vitamin A deficiency is common, high dose vitamin A supplementation of lactating mothers is currently recommended, but the effects of this intervention have not been carefully evaluated. We conducted a randomized, double-blind trial in which 153 Indonesian mothers 1-3 wk postpartum received either a capsule containing 312 mumol of vitamin A as retinyl palmitate or a placebo. Mothers' serum retinol concentrations in the vitamin A group tended to be lower than in the placebo group at baseline but higher at 3 mo postpartum (1.39 vs. 1.24 mumol/L, P = 0.03) and 6 mo postpartum (1.23 vs. 1.08 mumol/L, P < 0.01). The milk retinol concentrations of the vitamin A group were higher than those of the placebo group by 0.48 to 1.18 mumol/L at 1-8 mo postpartum (P < 0.05). Among the infants at 6 mo of age, the prevalences of low serum retinol concentration (< 0.52 mumol/L) were 36 and 15% in the placebo and vitamin A groups, respectively (P < 0.005), and the prevalences of low vitamin A stores (assessed by relative dose response) were 23 and 10%, respectively (P < 0.03). High dose vitamin A supplementation of lactating mothers is an efficacious way to improve the vitamin A status of both mother and breast-fed infant.

Adolescent↗
PubMed1993

Anesthetic cutoff in cycloalkanemethanols. A test of current theories.

BACKGROUND: N-alkanols containing up to 12 carbons are anesthetic; however, those with more than 12 carbons are not. This phenomenon has been termed cutoff. Lipid disordering theories of anesthesia suggest that cutoff occurs because the alkyl chains of long-chain alcohols approach the length and shape of the lipids of neuronal membranes and, therefore, intercalate into membranes without perturbing them. Protein theories suggest that cutoff occurs because the size of long-chain alcohols exceeds that of a protein binding site having finite dimensions. These theories were tested with a new series of alcohols, the cycloalkanemethanols, c(CnH2n-1).CH2.OH. METHODS: Anesthetic potency was measured in Rana pipiens tadpoles using the reversible loss of righting reflexes as the endpoint. The change in order parameter induced by cycloalkanemethanols and n-alkanols in lipid bilayers made of egg phosphatidylcholine and cholesterol was measured with electron paramagnetic resonance spectroscopy. RESULTS: On ascending the series from cyclopropanemethanol (EC50 = 54 +/- 3.2 mM) to cycloundecanemethanol (EC50 = 7.0 +/- 0.12 microM) anesthetic potencies first increased exponentially but then decreased sharply at cyclododecanemethanol (EC50 = 13 +/- 0.2 microM). Cyclotetradecanemethanol was found not to cause anesthesia in tadpoles, even after 48 h of exposure, although saturated solutions shifted the dose-response curve of octanol from 66 +/- 2.6 to 47 +/- 2.8 microM. A linear loss in the ability to disorder lipid bilayers was observed on ascending both alcohol series such that cyclotetradecanemethanol and n-tridecanol actually increased bilayer order. CONCLUSIONS: Molecular length does not correlate with anesthetic cutoff in these two alcohol series. Cutoff is predicted by the ability of both series of alcohols to disorder lipid bilayers and correlates with their molecular volume.

Anesthesia↗