Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism

Alexey V Rossokhin; Irina N Sharonova; Julia V Bukanova; Sergey N Kolbaev; Vladimir G Skrebitsky

doi:10.1016/j.mcn.2014.10.001

Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism

Mol Cell Neurosci. 2014 Nov:63:72-82. doi: 10.1016/j.mcn.2014.10.001. Epub 2014 Oct 8.

Authors

Alexey V Rossokhin¹, Irina N Sharonova², Julia V Bukanova², Sergey N Kolbaev², Vladimir G Skrebitsky²

Affiliations

¹ Research Center of Neurology, Russian Academy of Medical Sciences, 105064 Moscow, Russia. Electronic address: alrossokhin@yahoo.com.
² Research Center of Neurology, Russian Academy of Medical Sciences, 105064 Moscow, Russia.

PMID: 25305478
DOI: 10.1016/j.mcn.2014.10.001

Abstract

GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of β-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block.

Keywords: GABA(A) receptor; Isolated neurons; Molecular modeling; Monte-Carlo energy minimization; Patch clamp; Penicillin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Action Potentials
Amino Acid Sequence
Animals
Binding Sites
Cells, Cultured
GABA-A Receptor Antagonists / pharmacology*
Molecular Docking Simulation*
Molecular Sequence Data
Penicillin G / pharmacology*
Protein Binding
Purkinje Cells / drug effects
Purkinje Cells / physiology
Rats
Rats, Wistar
Receptors, GABA-A / chemistry*
Receptors, GABA-A / metabolism

Substances

GABA-A Receptor Antagonists
Receptors, GABA-A
Penicillin G