Genetic inhibition of KV7/M-channels increases the excitability of bronchopulmonary vagal nociceptive nerves

Abstract

The KV7/M-channel, encoded by Kcnq2-5 genes, generates a “braking” potassium current (IM) and contributes to the regulation of neuronal excitability. We have previously reported that the vagal sensory nerves terminated in the mouse lungs expressed mainly Kcnq3 and Kcnq2, as well as the functional M-channels. Opening the M-channel with retigabine almost abolished the agonist-evoked action potential (AP) discharges in bronchopulmonary nociceptive C-fibers, and suppressed the irritant gases-induced, C-fiber-mediated coughing in awake freely moving mice. Inhibition of IM using XE991 slightly but consistently depolarized the nodose neurons. The use of XE991 to evaluate whether IM plays a role in regulating the activation of bronchopulmonary vagal sensory nerves was, however, limited due to its off-target effects on the fast Na+ current in these neurons. This study aimed to address this key issue using the genetic approach. We generated the sensory neuron-specific Kcnq2 and Kcnq3 double-KO mice (Kcnq2/3-KO) by crossing the Kcnq2/3fl/fl mice with the Pirt-driven Cre (Pirt-Cre) mice. Single-neuron RT-PCR assay showed that none of the 60 tested Kcnq2/3-KO nodose neurons (30 Trpv1-positive and 30 Trpv1-negative) expressed Kcnq2 or Kcnq3. Eliminating Kcnq2 and Kcnq3 did not increase Kcnq4 or Kcnq5 expression. IM, defined as the retigabine-enhanced and XE991-inhibited tail currents (elicited by repolarizing steps from -30 to -60 mV), was recorded in all 22 control neurons using perforated patch clamp technique, but only in 2 out of 14 Kcnq2/3-KO nodose neurons (probably due to Kcnq5) with a much lower IM density (0.4 and 2.1 pA/pF vs. 8.5±2.9 pA/pF in controls). These results confirmed an effective elimination of the dominant KV7 genes in the nodose neurons of our Kcnq2/3-KO mice. Genetic inhibition of Kcnq2/3 did not significantly alter the resting potential (-68.0±1.9, n=12 vs. -69.1±1.4 mV, n=13; p=0.644) or the minimal amount of currents to evoke a single AP (rheobase: 41.2±6.1 pA, n=12 vs. 48.1±6.2 pA, n=13; p=0.442) in capsaicin-sensitive nodose neurons. However, the nociceptive nodose neurons isolated from Kcnq2/3-KO mice fired more APs and at a higher frequency in response to a series of either step (50-500 pA, 600 ms) or ramp (50-800 pA, 1 second) depolarization compared to their littermate controls (P<0.001, two-way ANOVA). Extracellular recordings in ex-vivo vagally innervated trachea-lungs preparations also revealed that Kcnq2/3 deficiency led to enhanced AP discharges at the nerve endings of the bronchopulmonary C-fibers in response to the agonist of Gq-coupled protease-activated receptor 1 (3, 10 and 30 μM) applied to the mouse lungs via trachea (number of AP: 393±45, n=8 vs. 183±43, n=9; p=0.001 by two-way ANOVA). These results indicate that the M-channel may serve as an intrinsic inhibitory mechanism in the bronchopulmonary vagal nociceptive neurons by limiting the excessive repetitive AP firings. Johns Hopkins Blaustein Pain Research Funds Grant 80048814, and NIH R01 HL137807/R35 HL155671 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.