Abstract
BackgroundLarge-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset.MethodsMyometrial biopsies were obtained at hysterectomy (non-pregnant, NP), and at Caesarean section, at elective (pregnant not-in-labour, PNL) and intrapartum (pregnant in-labour, PL) procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets.ResultsRT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities.ConclusionsOur findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at labour onset, coupled to an increased proportion of Maxi-K channels expressing the 132 bp spliced exon, may be linked to decreased Maxi-K channel calcium and voltage sensitivity, thereby promoting enhanced uterine activity at the time of labour.
Highlights
Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis
Analysis of expression of alternatively spliced exons of the Maxi-K α subunit in human myometrium reverse transcriptase negative controls (RT-)PCR analysis of the Maxi-K α subunit gene, using primers designed to flank predicted splice sites, produced a variety of bands in samples of non-pregnant (NP), pregnant not-in-labour (PNL) and pregnant in-labour (PL) myometrium
Sequence analysis of these bands confirmed the presence of two alternatively spliced exons, both of which had previously been identified in human myometrium
Summary
Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. The regulatory mechanisms for uterine smooth muscle contractility during human pregnancy and labour are poorly understood. There is a substantial body of evidence indicating that alternate splicing of the maxi-K transcript plays a major role in regulating potassium channel conductance [7,15]. These data include evidence for splice variation effecting calcium and voltage sensitivity, surface expression, and sensitivity to protein phosphorylation of the maxi-K channel [16,17]; [18]. What initiates alternative splicing of the α subunit transcript is incompletely understood, there is evidence that expression of different alternatively spliced transcripts can be hormonally induced [20,21]
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