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The splice variants of TREK-1 act as dominant-negative suppressors of human uterine relaxation: Implications for preterm labor
AdvisorBuxton, Iain L.O.
Biochemistry and Molecular Biology
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Human myometrium plays a pivotal role during pregnancy remaining quiescent during fetal development while eventually producing the forceful contractions necessary for parturition. The complex mechanism of maintaining uterine quiescence and triggering contraction is unclear. Spontaneous or unexplained preterm labor (PTL) accounts for approximately 45% of preterm birth (PTB) which results in infant death every 30 seconds globally. Two-pore domain potassium channels (K2P) are an emerging family of ion channels; these K2P channels participate in the regulation of excitable cells through maintaining resting membrane potential by polarizing the plasma membrane. The aberrant regulation of K2P channels may switch the cells from a resting state to one of excitation. TREK-1, a member of the K2P channel family, has previously been shown to exhibit elevated expression in pregnant human myometrium during gestation. In this study, three splice variants of intact human TREK-1 were identified in myometrium. For the first time, five non-conducting splice variants of TREK-1 were identified in preterm laboring and non-laboring myometrium, and were named as SV-1 to SV-5 due to the number of deleted exons. The exon skipping contributes to heavily truncated proteins with defective TREK-1 channel properties. Quantitative real-time PCR provided evidence of elevated expression of SV-5 in preterm labor, while TREK-1 was down-regulated. The up-regulation of SV-1, SV-3 and SV-5 are seen in preterm non-laboring myometrium.Alternative splicing has been shown to generate variants that can interfere with TREK-1 channel activity. Each individual splice variant was determined to be dominant-negative suppressors of TREK-1. The cellular distribution of TREK-1 was altered by each splice variant, which caused reduced membrane TREK-1 expression. Electrophysiological experiments confirmed the direct interaction between TREK-1 and each splice variant in basic and intracellular acidic conditions. Individual splice variants inhibited TREK-1 channel activity by reducing current densities as well as membrane TREK-1 expression. While intracellular acidosis is associated with uterine relaxation as well as TREK-1 channel activation, the use of bicarbonate experimentally to activate TREK-1 could not rescue TREK-1 channels blocked by co-expression of variant channels. Hetero-dimerization of TREK-1 with splice variant subunits is the probable mechanistic basis for this inhibition. Our research has shown that individual splice variants have the ability to impair TREK-1 channel function, although only SV-5 was significantly up-regulated in patients that delivered due to preterm labor. The presence of all non-conducting splice variants and the reduced TREK-1 transcript in preterm patients may also correlate with preterm labor per se. The presence of splice variants may competitively inhibit normal TREK-1 monomers which puts the uterus at risk of not maintaining quiescence in the presence of contractile influence(s). New therapeutic targets and biomarkers for preterm labor are suggested by this research due to the finding of non-conductive splice variants as dominant-negative suppressors of TREK-1.