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Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor.

ABSTRACT: Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

SUBMITTER: Pineda RH

PROVIDER: S-EPMC6148548 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Altered expression and modulation of the two-pore-domain (K<sub>2P</sub>) mechanogated potassium channel TREK-1 in overactive human detrusor.

Pineda Ricardo H RH Nedumaran Balachandar B Hypolite Joseph J Pan Xiao-Qing XQ Wilson Shandra S Meacham Randall B RB Malykhina Anna P AP

American journal of physiology. Renal physiology 20170524 2

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K<sub>2P</sub>) channels. In the present study, we aimed to identify the changes in expression and f ...[more]

PMID: 28539337

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Project description:BackgroundPreviously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice.MethodsAdult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals.ResultsTREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice.ConclusionsOur results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the "mixed" voiding phenotype in TREK-1 KO mice.

Project description:BACKGROUND AND PURPOSE:TREK two-pore-domain potassium (K2P ) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI-530159. EXPERIMENTAL APPROACH:The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single-channel patch-clamp recordings from recombinant TREK channels. The expression of K2P 2.1 (TREK1), K2P 10.1 (TREK2) and K2P 4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. KEY RESULTS:For recombinant human TREK1 channels, GI-530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current-clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1 ?M resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential. CONCLUSIONS AND IMPLICATIONS:This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid in the development of novel analgesic agents. LINKED ARTICLES:This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Dataset Information

Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor.

Publications

Altered expression and modulation of the two-pore-domain (K<sub>2P</sub>) mechanogated potassium channel TREK-1 in overactive human detrusor.

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