Identification and functional characterisation ofpotassium channels in mouse GI tract

Abstract

Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) occur when coordinated smooth muscle contractile activity is disrupted. Although much is known about the ionic conductances responsible for GI smooth muscle contraction, comparably little is known about the underlying potassium (K+) channels responsible for suppressing smooth muscle contractility and promoting GI tract relaxation. Using a mouse model, quantitative real-time PCR was used to establish the expression profiles of 93 K+ channel α- and β- subunit genes in ileum and colon. Immunostaining of selected K2P channel proteins, informed from the qPCR, revealed distinctive expression patterns. Specifically, the mechano-gated K2P channel, mTREK-1, was exclusively localised to smooth muscle of ileum and colon whereas the expression of related TREK-2 and TRAAK channel proteins was limited to enteric neurons. To assess the contribution of these K+ channels to the regulation of longitudinal smooth muscle tone, ileum and colon tissues were exposed to a variety of K2P channels modulators including BL-1249, CDC and riluzole using an organ bath preparation. Activation of mechano-gated K2P channels resulted in significant relaxation of both KCl and CCh pre-contracted ileum and colon tissues and reduced the amplitude of spontaneous contractions. Relaxation occurred in the presence of TTX, indicating a direct smooth muscle action of the K2P modulators. Given their prominent role in determining GI tract smooth muscle contractility, mouse homologues of the mechano-gated subfamily of channels were investigated further. Cloned mouse TREK-1, TREK-2 and TRAAK channels exhibited similar biophysical properties to their human homologues, with comparable sensitivity to well-known K2P activators AA, CDC and BL-1249. In contrast to previous findings, I reveal the proposed TREK-1 blocker, spadin, to possess the pharmacological characteristics of an antagonist. Spadin specifically antagonised the activation of cloned TREK-1 channels by the polyunsaturated fatty acid, arachidonic acid (AA), but not by other pharmacological activators. The generation of a chimera with the spadin insensitive mTREK-2 channel indicated that the mechanism of action of spadin does not involve the Cterminus directly, and likely works via an allosteric mechanism distinct from the AA binding site. This study has provided a new insight into the potassium channel subtype expression profile and function in mouse intestine, and highlights the mechano-gated K2P channel TREK-1 as a potential target for future drug development treating hypermotility GI disorders.

Date of Award	Jan 2019
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Anthony Lewis (Supervisor) & James Brown (Supervisor)