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Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic

Pavel, Mahmud Arif PhD*,†; Chung, Hae-Won PhD*,†; Petersen, E. Nicholas PhD*,†; Hansen, Scott B. PhD*,†

doi: 10.1213/ANE.0000000000004216
Preclinical Pharmacology: Original Laboratory Research Report

BACKGROUND: Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K+ channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel’s C terminus.

METHODS: Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM).

RESULTS: We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis.

CONCLUSIONS: We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.

From the Departments of *Molecular Medicine

Neuroscience, The Scripps Research Institute, Jupiter, Florida.

Published ahead of print 4 March 2019.

Accepted for publication April 3, 2019.

Funding: This work was supported by a Director’s New Innovator Award to S.B.H. (1DP2NS087943-01) from the National Institutes of Health.

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Reprints will not be available from the authors.

Address correspondence to Scott B. Hansen, PhD, Department of Molecular Medicine, The Scripps Research Institute, 130 Scripps Way No. C236, Jupiter, FL 33458. Address e-mail to

Copyright © 2019 International Anesthesia Research Society
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