Lei Qi, Pan Xiao-Qing, Chang Shaohua, Malkowicz S Bruce, Guzzo Thomas J, Malykhina Anna P
Division of Urology, Department of Surgery, University of Pennsylvania, PA, USA.
Department of Surgery, Cooper University, NJ, USA.
J Physiol. 2014 Jul 15;592(14):3013-30. doi: 10.1113/jphysiol.2014.271718. Epub 2014 May 6.
The mechanisms of mechanosensitivity underlying the response of the human bladder to stretch are poorly understood. Animal data suggest that stretch-activated two-pore-domain (K2P) K(+) channels play a critical role in bladder relaxation during the filling phase. The objective of this study was to characterize the expression and function of stretch-activated K2P channels in the human bladder and to clarify their physiological role in bladder mechanosensitivity. Gene and protein analysis of the K2P channels TREK-1, TREK-2 and TRAAK in the human bladder revealed that TREK-1 is the predominantly expressed member of the mechano-gated subfamily of K2P channels. Immunohistochemical labelling of bladder wall identified higher levels of expression of TREK-1 in detrusor smooth muscle cells in comparison to bladder mucosa. Functional characterization and biophysical properties of the predominantly expressed member of the K2P family, the TREK-1 channel, were evaluated by in vitro organ bath studies and the patch-clamp technique. Electrophysiological recordings from single smooth muscle cells confirmed direct activation of TREK-1 channels by mechanical stretch and negative pressure applied to the cell membrane. Inhibition of TREK-1 channels in the human detrusor significantly delayed relaxation of the stretched bladder smooth muscle strips and triggered small-amplitude spontaneous contractions. Application of negative pressure to cell-attached patches (-20 mmHg) caused a 19-fold increase in the open probability (NPo) of human TREK-1 channels. l-Methionine (1 mm), a specific TREK-1 inhibitor, dramatically decreased the NPo of TREK-1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Subsequent addition of arachidonic acid (10 μm), a channel opener, increased the open probability of methionine-inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). The results of our study provide direct evidence that the response of the human detrusor to mechanical stretch is regulated by activation of mechano-gated TREK-1 channels. Impaired mechanosensation and mechanotransduction associated with the changes in stretch-activated K2P channels may underlie myogenic bladder dysfunction in humans.
人们对人类膀胱对牵张作出反应背后的机械敏感性机制了解甚少。动物数据表明,牵张激活的双孔结构域(K2P)钾通道在充盈期膀胱舒张过程中起关键作用。本研究的目的是描述人类膀胱中牵张激活的K2P通道的表达和功能,并阐明它们在膀胱机械敏感性中的生理作用。对人类膀胱中K2P通道TREK-1、TREK-2和TRAAK进行基因和蛋白质分析发现,TREK-1是K2P通道机械门控亚家族中主要表达的成员。膀胱壁的免疫组织化学标记显示,与膀胱黏膜相比,逼尿肌平滑肌细胞中TREK-1的表达水平更高。通过体外器官浴研究和膜片钳技术评估了K2P家族主要表达成员TREK-1通道的功能特性和生物物理特性。对单个平滑肌细胞的电生理记录证实,机械牵张和施加于细胞膜的负压可直接激活TREK-1通道。抑制人类逼尿肌中的TREK-1通道会显著延迟被牵张的膀胱平滑肌条的舒张,并引发小幅度的自发收缩。对细胞贴附膜片施加负压(-20 mmHg)会使人类TREK-1通道的开放概率(NPo)增加19倍。特异性TREK-1抑制剂L-蛋氨酸(1 mM)可使TREK-1通道的NPo从0.045±0.003显著降至0.008±0.001(n = 8,P≤0.01)。随后添加通道开放剂花生四烯酸(10 μM)可使蛋氨酸抑制的单位电流在0 mV时的开放概率增加至0.43±0.05(n = 9,P≤0.05)。我们的研究结果提供了直接证据,表明人类逼尿肌对机械牵张的反应受机械门控TREK-1通道激活的调节。与牵张激活的K2P通道变化相关的机械感觉和机械转导受损可能是人类肌源性膀胱功能障碍的基础。