Biomedical subjects

R N Cox

Publications and source records attributed to R N Cox.

At least 19 recordsLinked to original sources

PubMed1986

Functional domains of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is a multisubunit, membrane-spanning protein that contains a gated, cation-conducting channel. Our approach to the understanding of the function of this receptor in molecular terms has been to locate its functionally significant sites in the sequences of its subunits and in its three-dimensional structure. In addition, we have tried to correlate transitions in the properties of these sites with functional transitions of the receptor. On binding acetylcholine, the nicotinic acetylcholine receptor enters at least two transient states, the open state and the rapid-onset desensitized state, and, in the continued presence of agonist, finally subsides into the slow-onset desensitized state. The transitions of the receptor between these various states are susceptible to regulation by acetylcholine and its congeners acting at one type of site and by a broad class of noncompetitive inhibitors (NCIs), including local anesthetics, acting at other sites. The chain composition of the receptor is alpha 2 beta gamma delta. The two acetylcholine binding sites are on the alpha chains, and two residues contributing to these sites, Cys-192 and Cys-193, have been identified. Furthermore, these adjacent Cys residues are cross-linked by a disulfide bond. In the quaternary structure of the receptor, the chains appear to be arranged in the order alpha gamma alpha beta delta around a central channel. Both the alpha and beta chains contribute to functionally significant NCI binding sites. The addition to receptor-rich membrane from Torpedo electric tissue of agonists (but not competitive antagonists) renders these NCI sites susceptible to photolabeling by the NCI quinacrine azide (QA). Furthermore, this susceptibility is transient, arising in milliseconds and subsiding in hundreds of milliseconds. These transiently susceptible sites are protected by other NCIs against photolabeling by QA. The time-course of the susceptibility and its dependence on agonist-concentration suggest that it might be the transient, rapid-onset desensitized state of the receptor that is most susceptible to photolabeling by QA.

Acetylcholine↗

PubMed1985

Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state.

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.

Affinity Labels↗

PubMed1984

The role of Ca2+ in cross-bridge kinetics in chemically skinned rabbit psoas fibers.

The sinusoidal analysis method is applied to chemically skinned rabbit psoas preparations, and the effect of Ca ions on exponential processes is determined. The results are compared to the predictions of two classes of models, those in which the binding of Ca2+ to the thin filaments acts as a switch to fully activate a segment of the filament (switch hypothesis), and those in which the binding of Ca2+ results in a graded increase in activity in proportion to the fraction of sites occupied (graded hypothesis). Although qualitative features of our results are in accord with the switch hypothesis, quantitative examination of the results shows that there is an extra process (B'), which produces a W-shaped phase-frequency plot during partial activation. This result is consistent with the assumption that there are two activated states available to the thin filaments of structured muscle.

Adenosine Triphosphatases↗

PubMed1984

A continuous-flow, rapid-mixing, photolabeling technique applied to the acetylcholine receptor.

A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.

Acetylcholine↗

PubMed1982

Effect of cross-bridge kinetics on apparent Ca2+ sensitivity.

Three different ways of shifting the pCa/tension curve on the pCa axis have been studied and related to changes in the rate constants of the cross-bridge cycle. The curve midpoint shifts to higher pCa's when the substrate (Mg-ATP) is reduced from 5 to 0.25 mM, when the phosphate concentration is reduced from 7.5 mM to 0, and when the ionic strength is reduced from 0.200 to 0.120. The Hill coefficients of the pCa/tension curve in our standard saline (5 mM substrate, 5 mM free ATP, 7.5 mM phosphate, ionic strength 0.200, 15 degree C) are between 5.1 and 5.6 and fall to 3.0 with the left shift of the curve brought about by reducing both substrate and phosphate. Left shifts of the curve produced by reduction in the ionic strength do not result ina lower Hill coefficient. Reducing eigher substrate or phosphate is associated with a reduction in the optimal frequency for oscillatory work, but reduction in ionic strength is not so associated. Maximum tension increases with the left shift of the curve brought about by reducing phosphate concentration or ionic strength, but tension decreases with the left shift of the curve accompanying substrate concentration reduction in phosphate-free saline. We argue that one mechanism for the observed shift of the curve along the pCa axis is the relationship between the time a cross-bridge takes to complete a cycle and the time Ca2+ stays bound to troponin C (TnC). If the cycle rate is decreased, a smaller fraction to TnC sites must be occupied to keep a given fraction of cross-bridges active. To illustrate this concept, we present a simplified model of the cross-bridge cycle incorporating the kinetics of Ca binding to TnC.

Adenosine Triphosphate↗

PubMed1981

Alternate energy transduction routes in chemically skinned rabbit psoas muscle fibres: a further study of the effect of MgATP over a wide concentration range.

Complex stiffness data were studied over an extended range of MgATP concentrations (3 muM-5 nM) in single fibres of Ca2+-activated, chemically skinned adult rabbit psoas. The data were analysed in terms of a model involving three exponential processes, the presence of which was previously observed in fully activated muscles. As fibres were transferred from a rigor solution into solutions of gradually increasing MgATP concentration, the three processes appeared sequentially, each with a unique Km. The order of appearance as MgATP increase is (1) the slowest of three processes [designated process (A)], (2) the fastest of the three processes [designated (C)], and (3) process (B), which occupies the middle range of frequencies; the KmS are approximately 10 muM, 0.2 mM, and 0.8 mM, respectively. The single phase advance [process (A)] remaining at very low substrate concentrations was found to be better described by a distribution of rate constants than by a single rate constant. The influence of substrate concentration on these processes is examined and two parallel hydrolysis routes are suggested as a possible mechanism.

Adenosine Triphosphate↗

PubMed1981

Effect of Ca ion concentration on cross-bridge kinetics in rabbit psoas fibers. Evidence for the presence of two Ca-activated states of thin filament.

The effect of Ca ion concentration on cross-bridge kinetics in a small bundle (one to three fibers) of chemically skinned rabbit psoas muscle is studied. The length of the muscle is oscillated in small amplitude sine waves (0.2% L0 peak-to-peak) at varying frequencies (0.125 -- 167 Hz), and the resulting amplitude and phase shift in tension are measured. The frequency response function (complex stiffness) thus obtained can be divided into three parts, which we name process (A) (centered at 1 Hz), process (B) (3--17 Hz), and process (C) (50 Hz). Process (B), which represents oscillatory work, further splits into two processes (B' and B) at partial Ca activation (less than 50% P0), where the phase-frequency plot appears W-shaped. The slower of the two processes (B') disappears by full activation, at which time the plot appears V-shaped. The characteristic frequencies associated with the minima of the plot do not shift in a graded way with Ca concentration, indicating that there is no change in apparent rate constants. Apparent rate constants of processes (A) and (C) are minimally affected by Ca. The above results are not altered when ionic strength is changed between 128 and 265 mM. We propose that activated thin filaments can have two "on" states and that Ca concentration controls the distribution of these two states. This mechanism generally supports the "switch" hypothesis of Ca regulation.

Animals↗

PubMed1980

Can the binding of Ca2+ to two regulatory sites on troponin C determine the steep pCa/tension relationship of skeletal muscle?

The relationship between tension and Ca2+ concentration in single skinned muscle fibers has been determined with a high density of experimental points and the data have been fitted by a least squares method to the Hill equation. We find that the mean Hill coefficient for the slope of the tension/Ca2+ relationship is between 5 and 6, and the pKd is about 5.9. Because there are four Ca2+ binding sites on troponin C, and only two of these regulate hydrolysis of MgATP, we conclude that the regulation of tension by Ca2+ binding is greatly modified by other factors. One important factor is the time required for a cross-bridge to complete a cycle once initiated, relative to the time Ca2+ remains bound to troponin C. The pCa/tension relationship will shift to higher pCa values as the ratio of cross-bridge cycle time to the Ca2+ bound time increases. For example, the pCa/tension curve may progressively shift to the left with increase in tension because strain in the myofilament lattice progressively increases the cycle time. This left shift will produce a pCa/tension relationship that is steeper than the actual Ca2+ binding curve. The anticipated shift of the pCa/tension curve with cycle time also bears on interpretations of earlier experiments on the "active state" and on the effects of Ca2+ on the maximal velocity of shortening.

Actins↗

PubMed1979

Activation of the frog sartorius acetylcholine receptor by a covalently attached group.

The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester of p-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor in Electrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorious motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic band.

Acetylcholine↗

PubMed1979

Voltage fluctuations at the frog sartorius motor endplate produced by a covalently attached activator.

The depolarization that develops after covalent attachment of trimethylammonium benzoyl to the dithiothreitol-reduced frog sartorius acetylcholine receptor is accompanied by a small increase in voltage fluctuations. The amplitude of the elementary voltage event produced by the covalently attached activator is about 0.04 microV, almost an order of magnitude below the acetylcholine shot-effect amplitude in the control preparation, and about one-fourth the acetylcholine shot amplitude after disulfide-bond reduction. Spectral density plots of trimethylammonium-benzoyl noise can be analyzed in terms of two relaxation rates that bracket the single rate observed in response to acetylcholine.

Acetylcholine↗

PubMed1974