Article Information

• PDF (924 kb)

PubMed

• Articles by R.L. Papke
• Articles by R.E. Oswald

Related Articles

• Open Channel Block and Alteration of N-Methyl-d-Aspartic Aci...

  Open Channel Block and Alteration of N-Methyl-d-Aspartic Acid Receptor Gating by an Analog of Phencyclidine Biophysical Journal, Volume 75, Issue 4, 1 October 1998, Pages 1801-1816 J.G. Dilmore and J.W. Johnson Abstract We investigated inhibition of the -methyl--aspartic acid (NMDA) receptor-channel complex by -ethyl-1,4,9,9-tetrahydro-4R-cis-4H-fluoren-4-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC of 0.51M at −66mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate () of 39.9Ms. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel. Abstract | Full Text | PDF (252 kb)

• Unbinding Pathways of an Agonist and an Antagonist from the ...

  Unbinding Pathways of an Agonist and an Antagonist from the 5-HT3 Receptor Biophysical Journal, Volume 90, Issue 6, 15 March 2006, Pages 1979-1991 A.J. Thompson, P.-L. Chau, S.L. Chan and S.C.R. Lummis Abstract The binding sites of 5-HT and other Cys-loop receptors have been extensively studied, but there are no data on the entry and exit routes of ligands for these sites. Here we have used molecular dynamics simulations to predict the pathway for agonists and antagonists exiting from the 5-HT receptor binding site. The data suggest that the unbinding pathway follows a tunnel at the interface of two subunits, which is ∼8Å long and terminates ∼20Å above the membrane. The exit routes for an agonist (5-HT) and an antagonist (granisetron) were similar, with trajectories toward the membrane and outward from the ligand binding site. 5-HT appears to form many hydrogen bonds with residues in the unbinding pathway, and experiments show that mutating these residues significantly affects function. The location of the pathway is also supported by docking studies of granisetron, which show a potential binding site for granisetron on the unbinding route. We propose that leaving the binding pocket along this tunnel places the ligands close to the membrane and prevents their immediate reentry into the binding pocket. We anticipate similar exit pathways for other members of the Cys-loop receptor family. Abstract | Full Text | PDF (660 kb)

• Extracellular Proton-Modulated Pore-Blocking Effect of the A...

  Extracellular Proton-Modulated Pore-Blocking Effect of the Anticonvulsant Felbamate on NMDA Channels Biophysical Journal, Volume 93, Issue 6, 15 September 2007, Pages 1981-1992 Huai-Ren Chang and Chung-Chin Kuo Abstract Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect is chiefly related to gating modification (and thus use-dependent inhibition) rather than pore block of -methyl--aspartate (NMDA) channels at pH 7.4. Using whole-cell recording in rat hippocampal neurons, we examined the effect of extracellular pH on FBM action. In sharp contrast to the findings at pH 7.4, the inhibitory effect of FBM on NMDA currents shows much weakened use-dependence at pH 8.4. Moreover, FBM neither accelerates the activation kinetics of the NMDA channel, nor enhances the currents elicited by very low concentrations of NMDA at pH 8.4. These differential effects of FBM between pH 7.4 and 8.4 are abolished in the mutant NMDA channels which lack proton sensitivity. Most interestingly, the inhibitory effect of FBM becomes flow-dependent and is evidently stronger in inward than in outward NMDA currents at pH 8.4. These findings indicate that FBM has a significantly more manifest pore-blocking effect on the NMDA channel at pH 8.4 than at pH 7.4. FBM therefore acts as an opportunistic pore blocker modulated by extracellular proton, suggesting that the FBM binding site is located at the junction of a widened and a narrow part of the ion conduction pathway. Also, we find that the inhibitory effect of FBM on NMDA currents is antagonized by external but not internal Na, and that increase of external Na decreases the binding rate without altering the unbinding rate of FBM. These findings indicate that the FBM binding site faces the extracellular rather than the intracellular solution, and coincides with the outmost ionic (e.g., Na) site in the NMDA channel pore. We conclude that the FBM binding site very likely is located in the external pore mouth, where extracellular proton, Na, FBM, and NMDA channel gating have an orchestrating effect. Abstract | Full Text | PDF (272 kb)

• …more

Abstract

The effects of the systematic variations of the acetylcholine molecule on the microscopic kinetics of channel activation were studied using the patch clamp technique. The modifications consisted of adding either halogens or a methyl group to the acetyl carbon of acetylcholine, which results in a change in both the steric and ionic character of that portion of the molecule. The ionic character of the bond affected both the opening and closing rates of the channel. An increase in the ionicity decreased the opening rate and increased the closing rate of the channel, suggesting that the open state was destabilized. Increasing the size of the substituent decreased both the association and dissociation rates for agonist binding but had little effect on the equilibrium constant. This indicates that the energy barrier for binding and unbinding was increased without a major change in the energy of the bound and unbound states. These results suggest that it is possible to assign changes in the structural characteristics of the ligand to changes in individual steps in a reaction scheme, which can lead to specific predictions for the properties of related compounds.