KCNQ Potassium Channels Reverse Opioid Suppression of Respiratory Drive in preBötzinger Dbx1+ Inspiratory Neurons by a Presynaptic Mechanism
Authors: Wei Aguan Daniel , Ramirez Jan‐Marino 
Published: November 2023

Reduced respiratory drive is a major morbidity risk from opioid‐based analgesics and drugs of abuse. Identifying molecular mechanisms that modulate opioid‐mediated suppression of respiratory drive may provide novel targets for pharmaceutically reversing opioid‐mediated respiratory suppression (ORS), without compromising analgesic efficacy. Although several brainstem sites likely contribute to ORS, we focused on the inspiratory central pattern generator (CPG) in the preBötzinger Complex (preBötC), best demonstrated to be essential for in vivo respiratory rhythmogenesis.We combined pharmacological and genetic approaches to examine ORS in mice (P7‐P13), by extracellular population and patch‐clamp recordings from in vitro brain slices that spontaneously generate rhythmic inspiratory bursts. ORS was studied by bath application of DAMGO, a mu‐opioid receptor (MOR1) specific agonist, which suppressed inspiratory frequency with an IC50 of 30 nM. MOR1 activation is known to inhibit neuronal excitability by signaling through the intracellular Gi/o pathway to activate GIRK potassium channels, and to suppress presynaptic P/Q‐(CaV2.1) and N‐type (CaV2.2) voltage‐gated calcium channels. Contrary to expectation, in vitro ORS was unaffected by a GIRK‐specific blocker (tertiapinQ), suggesting a minimal role for GIRK activation. However, genetic removal of CaV2.1 (CACNA1) sensitized the preBötC inspiratory pattern generator to DAMGO inhibition. Heterozygous CaV2.1 KO/+ slices were ~10‐fold more sensitive to DAMGO inhibition than WT, and homozygous CaV2.1 KO slices were ~5‐fold further shifted in sensitivity compared to heterozygous slices to DAMGO inhibition. In addition, we identified KCNQ potassium channels as novel modulators of in vitro ORS. Bath application of KCNQ‐specific inhibitors (Chromanol 293B, XE991) reversed, and a KCNQ activator (retigabine) mimicked DAMGO‐mediated in vitro ORS. Dose‐response profiles of these drug effects implicate KCNQ5 as the major KCNQ subtype underlying this modulatory effect. To test for a presynaptic site of action, miniature excitatory post‐synaptic currents (mEPSCs) were recorded from genetically‐labeled Dbx1+ inspiratory preBötC interneurons expressing tdTomato, in the presence of TTX. DAMGO suppressed excitatory mEPSC frequency significantly in 70% (5/7) of recorded neurons, and subsequent addition of XE991 restored mEPSC frequency in 83% (5/6) neurons. Thus, ORS within the preBötC acts through MOR1‐mediated suppression of presynaptic calcium channel function and excitatory transmitter release, which can be restored by blocking presynaptic KCNQ5 channels. ORS suppression of the inspiratory CPG likely results from a general dissociation of network function by degraded synaptic transmission below a critical quorum (or consistency) required for bursting, rather than inhibited intrinsic excitability in specific neurons. We have generated CRISPR‐targeted KCNQ5 KO mouse lines to further explore these ideas.Support or Funding InformationSupported by Seed Funds from Seattle Children's Research Institute and NIH R01‐HL126523‐01 (JMR).