title: Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women.
abstract: Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.
authors.affiliation: Department of Obstetrics and Gynecology, Basic Science Division, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Reinl
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52442, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Cabeza
authors.affiliation: Department of Obstetrics and Gynecology, Basic Science Division, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Ismail A
authors.initials: IA
authors.lastname: Gregory
authors.affiliation: Department of Obstetrics and Gynecology, Basic Science Division, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Alison G
authors.initials: AG
authors.lastname: Cahill
authors.affiliation: Department of Obstetrics and Gynecology, Basic Science Division, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA englands@wustl.edu.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: England
mesh_terms: D000328:Adult; D001706:Biopsy; D005260:Female; D005682:Gadolinium / Q000494:pharmacology; D020869:Gene Expression Profiling; D006801:Humans; D007473:Ion Channels; D017136:Ion Transport / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins; D009215:Myometrium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011247:Pregnancy; D011263:Pregnancy Trimester, Third / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014590:Uterine Contraction / Q000502:physiology*
chemical_list: D007473:Ion Channels; D008565:Membrane Proteins; C544681:NALCN protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D012964:Sodium; D005682:Gadolinium
keywords: contraction; leak current; myometrium; pregnancy; uterus