Inhibition of Human Two-Pore Domain K+ Channel TREK1 by Local Anesthetic Lidocaine: Negative Cooperativity and Half-of-Sites Saturation Kinetics

• Published in: Molecular pharmacology 2009-10, Vol.76 (4), p.903-917
• Main Authors: Nayak, Tapan K, Harinath, S, Nama, S, Somasundaram, K, Sikdar, S K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anesthetics, Local - administration & dosage; Anesthetics, Local - pharmacology; Cell Line; Dimerization; Humans; Kinetics; Lidocaine - administration & dosage; Lidocaine - pharmacology; Molecular Sequence Data; Potassium Channels, Tandem Pore Domain - antagonists & inhibitors; Potassium Channels, Tandem Pore Domain - chemistry; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.109.056838

TWIK-related K + channel TREK1, a background leak K + channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h)TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC 50 value of 180 μM, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Δ119 C-terminal deletion mutant (hTREK1 wt -Δ119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.