Modulated Receptor Hypothesis Research Articles

Inactivation induced by pathogenic Cav1.3 L-type Ca2+-channel variants enhances sensitivity for dihydropyridine Ca2+ channel blockers.

Pathogenic gain-of-function mutations in Cav1.3 L-type voltage-gated Ca2+-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+-channel blocker isradipine, a potential therapeutic for affected patients. Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.

Effect of phenytoin on sodium conductances in rat hippocampal CA1 pyramidal neurons.

The antiepileptic drug phenytoin (PHT) is thought to reduce the excitability of neural tissue by stabilizing sodium channels (NaV) in inactivated states. It has been suggested the fast-inactivated state (IF) is the main target, although slow inactivation (IS) has also been implicated. Other studies on local anesthetics with similar effects on sodium channels have implicated the NaV voltage sensor interactions. In this study, we reexamined the effect of PHT in both equilibrium and dynamic transitions between fast and slower forms of inactivation in rat hippocampal CA1 pyramidal neurons. The effects of PHT were observed on fast and slow inactivation processes, as well as on another identified "intermediate" inactivation process. The effect of enzymatic removal of IF was also studied, as well as effects on the residual persistent sodium current (INaP). A computational model based on a gating charge interaction was derived that reproduced a range of PHT effects on NaV equilibrium and state transitions. No effect of PHT on IF was observed; rather, PHT appeared to facilitate the occupancy of other closed states, either through enhancement of slow inactivation or through formation of analogous drug-bound states. The overall significance of these observations is that our data are inconsistent with the commonly held view that the archetypal NaV channel inhibitor PHT stabilizes fast inactivation states, and we demonstrate that conventional slow activation "IS" and the more recently identified intermediate-duration inactivation process "II" are the primary functional targets of PHT. In addition, we show that the traditional explanatory frameworks based on the "modulated receptor hypothesis" can be substituted by simple, physiologically plausible interactions with voltage sensors. Additionally, INaP was not preferentially inhibited compared with peak INa at short latencies (50 ms) by PHT.

Use-Dependent Block of Voltage-Gated Sodium Channels by Orphenadrine through Binding at the Local Anesthetic Receptor

Orphenadrine is most often used against muscle spasm and rigidity, and pain of various etiologies. The drug may act on multiple targets, including muscarinic, histaminic, and NMDA receptors. Its structure recalls that of local anesthetics, with an aromatic hydrophobic moiety linked to a protonable tertiary ammine terminal. Thus we wondered whether orphenadrine may also block voltage-gated sodium channels. We tested the drug on whole-cell sodium currents recorded using patch-clamp in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels. The results indicate that orphenadrine inhibits sodium channels in a concentration, voltage and frequency dependent manner. Introducing the F1586C mutation in hNav1.4 reduced two-fold the tonic block at the holding potential (hp) of -120 mV and almost zeroed the use-dependent block at 10 Hz, indicating that orphenadrine binds to the same receptor as the local anesthetics. Channel state-dependent affinities of orphenadrine were calculated using specific protocols. At the hp of -180 mV, the entire population of channels are closed and ready to open in response to depolarization. Thus determination of tonic block at this hp allows the determination of drug affinity for resting channels, which was 160 μM. The affinity for inactivated sodium channels (KI) was calculated according to the modulated receptor hypothesis, which forecasts that the apparent affinity measured at a particular hp depends on the proportion of resting and inactivated channels at this hp. The calculated KI value was 2.2 μM. Use-dependent reduction of sodium currents at the hp of -100 mV was enhanced by orphenadrine at 0.1 μM, a clinically relevant concentration. This study suggests that blockade of sodium channels may contribute to clinical efficacy of orphenadrine (Supported by Telethon-Italy grant GGP04140).

Exploring the heterogeneity of use-dependent sodium channel inhibitor drugs. II: Drugs described by the modulated receptor hypothesis (MRH) and/or the guarded receptor hypothesis (GRH)

Exploring the heterogeneity of use-dependent sodium channel inhibitor drugs. II: Drugs described by the modulated receptor hypothesis (MRH) and/or the guarded receptor hypothesis (GRH)

Setting up for the block: the mechanism underlying lidocaine's use‐dependent inhibition of sodium channels

Setting up for the block: the mechanism underlying lidocaine's use‐dependent inhibition of sodium channels

Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues.

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6–8.0, ∼50% neutral at pH 7.4) have similar structures but markedly different effects on Na+ channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na+ channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na+ channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na+ channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na+ channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.

Enhancing effects of salicylate on tonic and phasic block of Na + channels by class 1 antiarrhythmic agents in the ventricular myocytes and the guinea pig papillary muscle

Objective: To study the interaction between salicylate and class 1 antiarrhythmic agents. Methods: The effects of salicylate on class 1 antiarrhythmic agent-induced tonic and phasic block of the Na + current ( I Na) of ventricular myocytes and the upstroke velocity of the action potential ( V max) of papillary muscles were examined by both the patch clamp technique and conventional microelectrode techniques. Results: Salicylate enhanced quinidine-induced tonic and phasic block of I Na at a holding potential of −100 mV but not at a holding potential of −140 mV; this enhancement was accompanied by a shift of the h ∞ curve in the presence of quinidine in a further hyperpolarized direction, although salicylate alone did not affect I Na. Salicylate enhanced the tonic and phasic block of V max induced by quinidine, aprindine and disopyramide but had little effect on that induced by procainamide or mexiletine; the enhancing effects were related to the liposolubility of the drugs. Conclusions: Salicylate enhanced tonic and phasic block of Na + channels induced by class 1 highly liposoluble antiarrhythmic agents. Based on the modulated receptor hypothesis, it is probable that this enhancement was mediated by an increase in the affinity of Na + channel blockers with high lipid solubility to the inactivated state channels.

A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated mu1 Na+ channels.

According to Hille's modulated receptor hypothesis, benzocaine shares a common receptor with all other local anesthetics (LAs) in the voltage-gated Na+ channel. We tested this single receptor hypothesis using mutant muscle Na+ channels of mu1-I1575A, F1579A, and N1584A transiently expressed in Hek-293t cells. Both benzocaine and etidocaine are more effective at blocking mu1-N1584A current than the wild-type current, while they are less potent at blocking mu1-F1579A current. Such concurrent changes of both benzocaine and etidocaine potency towards F1579A and N1584A mutants suggest that they share a common LA receptor. Consistent with results found in studies of native Na+ channels, permanently charged QX-314 at 1 mM is not effective at blocking wild-type, F1579A, and N1584A current via external application. In contrast, QX-314 is relatively potent at blocking I1575A current when applied externally. This increased potency of external QX-314 against the mu1-I1575A mutant has been reported previously in a study of the brain counterpart. Mutant I1575A also appears to be highly sensitive to the external divalent cation Cd2+, probably because of the presence of cysteine residues near the mu1-I1575 position in the IV-S6 segment. To our surprise, neutral benzocaine becomes more effective at blocking mu1-I1575A current than the wild-type current, whereas the opposite is found for etidocaine. We hypothesize that an increase in accessibility of external QX-314 to the mu1-I1575A mutant is accompanied by a reduction of binding towards the charged amine component.

Stereoselectivity of Ca 2+ channel block by dihydropyridines: no modulation by the voltage protocol

The L-type Ca 2+ current inhibition by the enantiomers of the dihydropyridine niguldipine was investigated at various holding potentials (−40 to −120 mV) and stimulus frequencies (0.1–1 Hz), using guinea-pig ventricular myocytes. Block of whole-cell current is both voltage- and concentration-dependent. ( S)-Niguldipine is more potent than its ( R)-enantiomer. However, the extent of enantioselectivity is rather small (≤ × 4.4). Importantly, this value does not increase when stimulus conditions favour the inactivated channel state, although this leads to more potent block. This is in contrast to our expectation based on modulated receptor hypothesis, and to the high enantioselectivity of niguldipine binding found in guinea-pig heart membranes (× 40). We conclude that the common modulated receptor hypothesis has to be refined to explain the effects of niguldipine enantiomers.

Classification and mechanism of action of antiarrhythmic drugs.

The present paper reviews classification and mode of action of agents that suppress extrasystoles and tachyarrhythmias. These are classified according to their electrophysiological effects observed in isolated cardiac tissues in vitro (Vaughan Williams, 1989). Fast sodium channel blockers (class I) which reduce the upstroke velocity of the action potential are usually subclassified into three groups, class I A-C, according to their effect on the action potential duration. Beta-adrenergic antagonists (class II) exert their effects by antagonizing the electrophysiological effects of beta-adrenergic catecholamines. Class III antiarrhythmic agents (eg amiodarone) prolong the action potential and slow calcium channel blockers (class IV) suppress the calcium inward current and calcium-dependent action potentials. The classification of antiarrhythmic drugs is still under debate. This particularly applies to agents of class I and III. The effect of class I agents is frequency-dependent because the binding affinity of these drugs to the sodium channel is modulated by the state of the channel (modulated receptor hypothesis). Class I agents bind to the channel in the activated and inactivated state and dissociate from the channel in the rested state. This occurs at a drug-specific rate so that class I agents can be subclassified into only two groups, namely in those of the slow- and fast-recovery type respectively (time constant of reactivation greater or smaller than 1 s). Slow-recovery class I agents affect regular action potentials at normal heart rates which can more easily lead to a lengthening of the QRS duration in the ECG, to conduction disturbances and hence to pro-arrhythmic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

The modulated receptor hypothesis revisited from the viewpoint of myocardial interstitial potential.

Time- and voltage-dependent interaction of antiarrhythmic agents with target cardiac ion channels is termed the modulated receptor hypothesis. Actually class I agents suppress the maximum upstroke rate (Vmax) of intracellular potential (Vic) depending on the pacing cycle length (PCL) and external potassium concentration ([K+]e). We examined this concept from the aspect of interstitial potential (Vis), since Vis reflects the second time derivative of Vic. Vic and Vis were recorded sequentially using standard microelectrode applied to the paced and superfused guinea pig papillary muscles. In the steady state, the greatest negative deflection of Vis (Vmin) was suppressed by quinidine (10 microM) in both PCL- and [K+]e-dependent manner, just like Vmax. However, quinidine-induced greater inhibition of Vmin than Vmax was evident at shorter PCL and greater [K+]e. Based on the sequential alteration of PCL and exposure to ouabain (10 microM), different quinidine sensitivity between Vmax and Vmin is most likely accounted for by the activity-dependent K+ efflux and Na(+)-K+ pump-mediated K+ uptake (i.e., [K+]e fluctuation). Thus, the modulated receptor hypothesis is concluded to be valid in terms of Vis.

Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study.

The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage-dependence of gating, preventing full movement of the voltage sensor.

Quantitative structure—activity relationship study of antiarrhythmic phenylpyridines using multivariate partial least squares modelling

Abstract The partial least squares (PLS) method was applied to a study of antiarrhythmic activity data of phenylpyridines. A two-component PLS model based on three physicochemical parameters successfully separated active from inactive compounds. Since the modelling power of each physicochemical parameter is great enough, these coefficients of the PLS model equation are interpreted as measures of the contribution of physicochemical parameters. From the PLS discriminant function, a more potent antiarrhythmic agent can be produced by increasing the molar refractivity value of a substituent R on the benzene ring, increasing the torsion angle between amide function and the benzene ring and decreasing the proton affinity of an amino group of a phenylpyridine. The PLS model equation corresponds to the modulated-receptor hypothesis proposed by Hille as a mechanism for the antiarrhythmic agent-action for sodium channels. The PLS method has proved to be more predictive and promising in multivariate statistical methods for quantitative structure-activity relationship analysis than a linear learning machine approach.

Class III antiarrhythmic action of d-sotalol during hypothermia

To investigate whether changes in temperature influence the electrophysiologic effects of the class III antiarrhythmic agent d-sotalol, we studied its effects on propranolol-pretreated guinea pig papillary muscles at temperatures ranging from 37° to 27° C by means of conventional microelectrode techniques. We also examined the rate-dependent effect of d-sotalol at 37° and 27° C. Before the addition of d-sotalol, reducing the temperature from 37° to 27° C increased the action potential duration recorded at 50% repolarization (APD 50) from 112 ± 7 msec to 271 ± 15 msec and action potential duration recorded at 90% repolarization (APD 90) from 136 ± 7 msec to 325 ± 10 msec. d-Sotalol (50 μmol/L) lengthened APD 50 and APD 90 to a greater degree at low temperatures. Thus at 37° C d-sotalol lengthened APD 50 and APD 90 by 12 ± 6 msec and 19 ± 5 msec, and at 27° C by 37 ± 5 msec and 52 ± 7 msec, respectively. d-Sotalol produced its greatest effect on APD at long pacing cycle lengths, thus demonstrating reverse dependence. This rate-dependent effect was more marked at 27° C than at 37° C. The greater effect of d-sotalol on APD at long pacing cycle lengths may be explained by the modulated receptor hypothesis, assuming that the drug has a higher affinity for closed potassium channels. Such a mechanism may also explain the accentuated class III antiarrhythmic action of d-sotalol observed during hypothermia.

QSAR ANALYSIS OF ANTIARRHYTHMIC PHENYLPYRIDINES USING PARTIAL LEAST SQUARES METHOD

Antiarrhythmic activity data of phenylpyridines were investigated by the PLS method. The PLS equation supported for the modulated-receptor hypothesis in Na channels on cardiac muscle cell. From the obtained PLS modelling equation, the potent antiarrhythmic agents can be produced by increasing the MR value of the substituent R on benzene ring, increasing the proton affinity(corresponding to pKa) of an amino group of a phernylpyridine.

Interaction of forskolin with voltage-gated K+ channels in PC12 cells

Forskolin (FSK) directly blocks a distinct class of voltage-dependent K+ channels in pheochromocytoma cells. We have studied the biophysical mechanism of FSK action on these channels. The mean open duration decreased linearly with [FSK], indicating that a single molecule of FSK interacts with a single open K+ channel. FSK did not alter the voltage dependence of activation or the latency to first opening. Whole-cell currents in the presence of FSK did not show a rising phase in tail currents, suggesting that FSK-bound channels can close. We used a kinetic scheme in which FSK binds preferentially to the open state of the channel to describe its interaction with the K+ channel. This scheme is analogous to the modulated receptor hypothesis used to describe the interaction of local anesthetics with voltage-dependent Na+ channels.

Use of ionic currents to identify and estimate parameters in models of channel blockade

Models of ion channel blockade are frequently validated with observations of ionic currents resulting from electrical or chemical stimulation. Model parameters for some models (modulated receptor hypothesis) cannot be uniquely determined from ionic currents. The time course of ionic currents reflects the activation (fraction of available channels that conduct in the presence of excitation) and availability of channels (the ability of the protein to make a transition to a conducting conformation and where this conformation is not complexed with a drug). In the presence of a channel blocking agent, the voltage dependence of availability appears modified and has been interpreted as evidence that drug-complexed channels exhibit modified transition rates between channel protein conformations. Because blockade and availability both modify ionic currents, their individual contributions to macroscopic conductance cannot be resolved from ionic currents except when constant affinity binding to a bindable site is assumed. Experimental studies of nimodipine block of calcium channels and lidocaine block of sodium channels illustrate these concepts.

Fast and slow blockade of sodium channels by flecainide in rabbit cardiac Purkinje fibres

The electrophysiological effects of flecainide were tested using the two-microelectrode voltage-clamp technique and Vmax-measurements in isolated rabbit cardiac Purkinje fibres. Flecainide predominantly unfolds its sodium-channel blocking action during the upstroke phase of the cardiac action potential, because its Vmax-depressant effects are independent of the duration of the depolarizing interval. Very long lasting depolarizations caused a second, very slow blocking activity. Starting from a steady-state block, recovery from block was tested and yielded a time constant of 7.3 s for a membrane potential of -105 mV. The strong blockade of sodium-channels combined with a delayed recovery behaviour of the drug-associated channels gives reasons for the observation of a marked use-dependent block. This block increased when the cycle length was shortened or the holding potential was less negative. Additional application of lidocaine in several concentrations did not significantly increase or attenuate the phasic block caused by flecainide alone. Under special conditions we investigated flecainide's depression and shift of the Vmax/Vm-relation and we observed that the concentration dependence of both parameters could be described by simple 1:1 binding reaction. The effects of flecainide are largely reversible often greater than or equal to 15 min. Flecainide could be characterized as an open channel blocker with a very slow inactivated channel blocking activity. For the qualitative description of the sodium-channel block by flecainide we used the "modulated-receptor hypothesis", whereas for reconstructions of the use-dependent action we applied the "guarded-receptor hypothesis", which enables computations of phasic block with the knowledge of only one forward and one reverse rate constant.