Modeling ion channel blockade at guarded binding sites: application to tertiary drugs

1986 ◽  
Vol 251 (4) ◽  
pp. H848-H856 ◽  
Author(s):  
C. F. Starmer ◽  
K. R. Courtney
Keyword(s):  
Binding Site ◽  
Sodium Current ◽  
Ph Dependence ◽  
Channel Blockade ◽  
Channel State ◽  
Channel Inactivation ◽  
Excitable Membranes ◽  
Channel Blocking ◽  
Apparent Variation ◽  
Modulated Receptor

Excitable membranes exposed to sodium channel blocking agents (D; local anesthetics and antiarrhythmic drugs) show a progressive reduction of peak sodium current when repetitively depolarized (use dependence). Thus, with repetitive excitation, use dependence reflects a net rightward shift in the balance between unblocked channels (U) and blocked channels (B): U + D in equilibrium with B. The modulated receptor hypothesis (a 7-parameter model) has been proposed to account for this shift and is based on a channel lumen binding site whose affinity varies with channel state and where drug-complexed channels exhibit modified inactivation gate kinetics. Alternatively, we consider use-dependent binding as the result of transient access to a constant-affinity binding site. In this setting, the channel gate conformation is viewed as controlling the flux of drug as it diffuses between drug pools and the binding site. Apparent variation in binding rates is therefore considered the result of variations in the fraction of accessible sites. This guarded receptor hypothesis, with three fewer parameters, is able to predict apparent changes in channel binding and apparent shifts in channel inactivation without incorporating modified gating parameters in drug-complexed channels. Furthermore, with this model one is able to characterize both relaxation kinetics and channel blockade associated with tertiary amines as well as hydrophobic and hydrophilic agents. The pH dependence of repriming rates is utilized to estimate several of the important parameters associated with this simplified hypothesis.

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

1990 ◽  
Vol 259 (2) ◽  
pp. H626-H634
Author(s):  
C. F. Starmer ◽  
V. V. Nesterenko ◽  
F. R. Gilliam ◽  
A. O. Grant
Keyword(s):  
Time Course ◽  
Experimental Studies ◽  
Ionic Currents ◽  
Blocking Agent ◽  
Model Parameters ◽  
Protein Conformations ◽  
Channel Blockade ◽  
Channel Blocking ◽  
Individual Contributions ◽  
Modulated Receptor

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.

Is there a second external lidocaine binding site on mammalian cardiac cells?

1989 ◽  
Vol 257 (1) ◽  
pp. H79-H84 ◽  
Author(s):  
L. A. Alpert ◽  
H. A. Fozzard ◽  
D. A. Hanck ◽  
J. C. Makielski
Keyword(s):  
Binding Site ◽  
Cardiac Arrhythmias ◽  
Local Anesthetics ◽  
Sodium Current ◽  
Cardiac Cells ◽  
Na Channel ◽  
Functional Difference ◽  
Na Channels ◽  
Internal Perfusion ◽  
Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

Ethacizin blockade of calcium channels: a test of the guarded receptor hypothesis

1989 ◽  
Vol 257 (5) ◽  
pp. H1693-H1704
Author(s):  
C. F. Starmer ◽  
A. I. Undrovinas ◽  
F. Scamps ◽  
G. Vassort ◽  
V. V. Nesterenko ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Cell Voltage ◽  
Membrane Potentials ◽  
Specific Rate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Channel Inactivation ◽  
Ventricular Cells ◽  
Channel Blocking ◽  
Voltage Dependent

The effect on calcium channels of the sodium channel antagonist, ethacizin, was studied in isolated frog ventricular cells using the whole cell voltage-clamp methodology. Ethacizin was found to block inward calcium current in a frequency-, voltage-, and concentration-dependent manner. The frequency-dependent blocking properties were modeled by considering the drug interaction with a voltage-dependent mixture of calcium channels harboring either an accessible or an inaccessible binding site. With repetitive stimulation, the pulse-to-pulse reduction in peak current is shown to be exponential, with a rate linearly related to the interstimulus interval and the drug concentration. Observed frequency- and concentration-dependent blocks were consistent with the predictions of the model, and mixture-specific rate constants were estimated from these data. The negligible shift in channel inactivation and the reduction of apparent binding and unbinding rates with more polarized membrane potentials imply the active moiety of ethacizin blocks open channels and is trapped within the channel at resting membrane potentials. The binding rate at 0 mV is similar to that observed in studies of interactions of other open channel blocking agents with voltage- and ligand-gated channels.

scholarly journals Interactions of neosaxitoxin with the sodium channel of the frog skeletal muscle fiber.

1991 ◽  
Vol 97 (3) ◽  
pp. 561-578 ◽  
Author(s):  
S L Hu ◽  
C Y Kao
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Relative Abundance ◽  
Sodium Current ◽  
Ph Dependence ◽  
Hydroxyl Group ◽  
Dimensional Structure ◽  
Hydroxyl Groups ◽  
Frog Skeletal Muscle ◽  
Relative Potencies

Neosaxitoxin (neoSTX) differs structurally from saxitoxin (STX) in that the hydrogen on N-1 is replaced by a hydroxyl group. On single frog skeletal muscle fibers in the vaseline-gap voltage clamp, the concentrations for reducing the maximum sodium current by 50% (ED50) at pH's 6.50, 7.25, and 8.25 are, respectively, 4.9, 5.1, and 8.9 nM for STX and 1.6, 2.7, and 17.2 nM for neoSTX. The relative potencies of STX at the different pH's closely parallel the relative abundance of the protonated form of the 7,8,9 guanidinium function, but the relative potencies of neoSTX at the same pH's vary with the relative abundance of the deprotonated N-1 group. In constant-ratio mixtures of the two toxins, the observed ED50's are consistent with the notion that the two toxins compete for the same site. At pH's 6.50 and 7.25, the best agreement between observed and computed values is obtained when the efficacy term (epsilon) for either toxin is 1. At pH 8.25 the best agreement is obtained if the efficacy is 1 for STX but 0.75 for neo-STX. The marked pH dependence of the actions of neoSTX probably reflects the presence of a site in the receptor that interacts with the N-1 -OH, in addition to those interacting with the 7,8,9 guanidinium and the C-12 hydroxyl groups. Considering the three-dimensional structure of the STX and neoSTX molecules, the various site points are probably located in a fold or a crevice of the channel protein, where the extracellular orifice of the sodium channel is located.

The dependence of the maximum rate of rise of the action potential upstroke on membrane properties

1981 ◽  
Vol 214 (1194) ◽  
pp. 85-98 ◽  
Keyword(s):  
Action Potential ◽  
Maximum Rate ◽  
Sodium Current ◽  
Membrane Properties ◽  
Fractional Change ◽  
Sodium Conductance ◽  
Excitable Membranes ◽  
Real Relation ◽  
Voltage Clamping ◽  
Maximum Rate Of Rise

The maximum rate of rise of the action potential ( V̇ max ) is often used to study the maximum sodium conductance ( Ḡ Na ) of excitable membranes, by assuming that V̇ max is proportional to Ḡ Na . However, the real relation between V̇ max and Ḡ Na is uncertain. We use simple analytical descriptions of the membrane currents to investigate this relation. If (1) the sodium conductance is much greater than the non-sodium conductance of the membrane, (2) the sodium current is activated extremely quickly, and (3) the sodium current is inactivated extremely slowly, then V̇ max will indeed be proportional to Ḡ Na . However, if conditions (1) or (3) are not satisfied, the V̇ max – Ḡ Na relation will be non-proportional, such that a certain fractional change of Ḡ Na produces a larger fractional change of V̇ max . If condition (2) is not satisfied the V̇ max – Ḡ Na relation is distorted in the opposite direction, such that a certain fractional change of Ḡ Na produces a smaller fractional change of V̇ max . Measurements of V̇ max are usually performed in preparations where voltage clamping cannot be used to study Ḡ Na directly. However, voltage clamping is necessary to verify that conditions (1)–(3) are satisfied. The results of studies using V̇ max alone as a measure of Ḡ Na should be assessed with caution.

scholarly journals An Epitope of the Semliki Forest Virus Fusion Protein Exposed during Virus-Membrane Fusion

1999 ◽  
Vol 73 (12) ◽  
pp. 10029-10039 ◽  
Author(s):  
Anna Ahn ◽  
Matthew R. Klimjack ◽  
Prodyot K. Chatterjee ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Binding Site ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Ph Dependence ◽  
Fusion Peptide ◽  
Endocytic Pathway ◽  
Spike Protein ◽  
Viral Fusion ◽  
Wide Range

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

scholarly journals A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition

1990 ◽  
Vol 266 (3) ◽  
pp. 645-651 ◽  
Author(s):  
W Templeton ◽  
D Kowlessur ◽  
E W Thomas ◽  
C M Topham ◽  
K Brocklehurst
Keyword(s):  
Transition State ◽  
Binding Site ◽  
Kinetic Data ◽  
Model Building ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Structural Basis ◽  
Compound I

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional properties of rat and human neocortical voltage-sensitive sodium currents

1994 ◽  
Vol 71 (3) ◽  
pp. 1052-1064 ◽  
Author(s):  
T. R. Cummins ◽  
Y. Xia ◽  
G. G. Haddad
Keyword(s):  
Binding Site ◽  
Current Density ◽  
Sodium Current ◽  
Neonatal Rat ◽  
Sodium Currents ◽  
Adult Human ◽  
Decay Component ◽  
Site Density ◽  
Neocortical Neurons ◽  
Steady State Inactivation

1. The functional properties of sodium currents in acutely dissociated adult human, neonatal rat [postnatal day (P) 3 and P10], and mature rat (P21-23) neocortical pyramidal neurons were studied using whole-cell patch-clamp techniques. 2. The voltage dependence of activation and steady-state inactivation of neonatal rat sodium currents was shifted in the positive direction when compared with mature rat sodium currents. In contrast, no difference was detected between the voltage dependence of activation and steady-state inactivation of mature rat and adult human sodium currents. 3. The fast inactivation of rat (neonatal and mature) and human neocortical sodium currents were best fit with three components; a fast decay component, a slow decay component, and a persistent component. The magnitude of the persistent current in neocortical neurons averaged 1-3% of the peak current. Inactivation was faster for sodium currents in neonatal rat neocortical neurons than in mature neurons. No difference was detected in the kinetics of inactivation between mature rat and adult human sodium currents. 4. Saxitoxin (STX) inhibited neuronal sodium currents at nanomolar concentrations in neonatal and mature rat and adult human neocortical neurons. STX-insensitive channels were not detected. 5. STX affinity was also assayed using 3H-STX. A single high-affinity binding site was found in neonatal rat, mature rat, and adult human neocortical tissue. A developmental increase in STX binding site density in the rat neocortex was tightly correlated with the increase in the sodium current density (normalized to cell capacitance). Human neocortical tissue and mature rat neocortical tissue did not differ in STX binding site density or sodium current density. 6. From these electrophysiological and autoradiographic studies we conclude that 1) the increase in the normalized sodium current density and STX binding density with age postnatally reflects an increase in binding sites of sodium channels functionally expressed on neuronal membranes, 2) the functional differences in channel behavior with maturation can explain the higher threshold for excitation in neonatal neocortical neurons and the increase in accommodation or adaptation in firing in the mature neuron, and 3) mature rat neocortical neurons represent a valid model for the study of adult human pyramidal neocortical neurons in terms of Na+ channel expression and function.

Sign in / Sign up

Export Citation Format

Share Document