TY - JOUR
T1 - Sodium leak channel, non-selective contributes to the leak current inhuman myometrial smooth muscle cells from pregnant women
AU - Reinl, Erin L.
AU - Cabeza, Rafael
AU - Gregory, Ismail A.
AU - Cahill, Alison G.
AU - England, Sarah K.
N1 - Publisher Copyright:
© The Author 2015.
PY - 2015/2/25
Y1 - 2015/2/25
N2 - Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca2+ influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, areknownto causemembrane depolarization in manytissue types. Previously, aGd3+-sensitive, Na+-dependent leak currentwas 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 Ca2+ 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 μMGd33+ 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 Gd3+-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.
AB - Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca2+ influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, areknownto causemembrane depolarization in manytissue types. Previously, aGd3+-sensitive, Na+-dependent leak currentwas 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 Ca2+ 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 μMGd33+ 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 Gd3+-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.
KW - Contraction
KW - Leak current
KW - Myometrium
KW - Pregnancy
KW - Uterus
UR - http://www.scopus.com/inward/record.url?scp=84943750444&partnerID=8YFLogxK
U2 - 10.1093/molehr/gav038
DO - 10.1093/molehr/gav038
M3 - Article
C2 - 26134120
AN - SCOPUS:84943750444
SN - 1360-9947
VL - 21
SP - 816
EP - 824
JO - Molecular human reproduction
JF - Molecular human reproduction
IS - 10
ER -