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

Abstract：

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.

journal_name

Anesth Analg

journal_title

Anesthesia and analgesia

authors

Pavel MA,Chung HW,Petersen EN,Hansen SB

doi

10.1213/ANE.0000000000004216

subject

Has Abstract

pub_date

2019-10-01 00:00:00

pages

973-982

issue

eissn

0003-2999

issn

1526-7598

journal_volume

129

pub_type

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