Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC349
The Novel Potassium Channel Kir7.1 is a Critical Component of Uterine Quiescence in Mice and Human.
C. McCloskey1, E. Bailey1, J. Zhang1, A. Shmygol1, S. Thornton1, R. D. Catalano2, S. England3, C. Rada3, A. M. Blanks1
1. CSRI, University of Warwick, Coventry, United Kingdom. 2. MRC HRSU, Queens Medical Research Institute, Edinburgh, United Kingdom. 3. Molecular Physiology & Biophysics, University of Iowa, Iowa, Iowa, United States.
A genome-wide screen of myometrial smooth muscle (MSM) mRNA expression revealed a novel channel (Kir7.1, KCNJ13) that displays biophysical properties suited to mediating uterine quiescence. Computer simulations predicted that Kir 7.1 could be a critical regulator of myometrial cell excitability. To address this, myometrial biopsies were taken in women at the time of C-Section (PTL 28-32 Wks, T 37-40wks NIL n=8, TLAB, n=8) and in mice GD13-18 (n=5/GD). qPCR was utilized to determine gestation dependent expression. Protein localisation was determined by immunohistochemistry. In vitro force recordings before and after administration of the inhibitor, VU590 determined the functional contribution of Kir7.1. Membrane potential (Vm) was determined by administration of VU590 under current clamp conditions using microelectrodes. In vivo contractility was tested by direct injection of VU590 to uterine horns of pregnant mice (n=3). In mice, KCNJ13 mRNA increased 30-fold (G13 vs GD15, P<0.001) to a peak GD15 followed by a 20-fold decline to term (GD15 vs GD18, P<0.001). In human TNIL and TLAB were >4-fold less when compared to PTNIL (Student's t-test: P=0.049 and 0.042 respectively). In mice and human, IHC demonstrated specific staining for MSM. Vu590 stimulated a dose dependent increase in tone as a percentage of control in both GD 15/18 mouse myometrium, this increase is significantly greater in GD15 than GD18, as would be expected due to the 20-fold increase in mRNA expression. OT elicited a significant increase in tone in GD 15 + 18 myometrium (438.1 ± 95.3% and 806.7 ± 153.8% respectively), however when OT was co administered with VU590 10 μM a substantially greater increase in tone was recorded (17294.4 ± 1457.0% and 8825.2 ± 4309.8%). Application of 100 μM VU590 to human myometrium stimulates an immediate contraction, typically > 20 min duration. Application of 1 μM VU590 to mice and human stimulated contraction followed by an enhancement of contractile force without the tonic component. Co-administration of 1nM OT and 30 μM VU590 elicited long lasting contractions of up to 20hrs. In current clamp experiments on GD15/18 mice, VU590 10 μM was found to stimulate a depolarisation to threshold and increase action potential frequency and duration. OT 1nM applied concomitantly with VU590 10 μM had a similar yet prolonged response. The effect of VU590 on Vm recorded from human myometrium was similar to that recorded in mouse, with depolarisation maintained until the drug was thoroughly washed out. Injection of 100 μM VU590 directly into the uteri of pregnant mice (GD15-18) induces an immediate and tonic contraction (n=3). In conclusion Kir7.1 is critical for uterine quiescence. Computer simulations predict a two-fold importance. Firstly, in maintaining Vm below threshold and secondly in mediating AP spiking frequency and hence force during a contraction.
Where applicable, experiments conform with Society ethical requirements