Research On The Selective Modulation Effects Of Fasudil, A Rho Kinase Inhibitor, On Kv7Potassium Channels

The Kv7channel family (Kv7.1-7.5), encoded by KCNQ genes, is afamily of voltage-dependent ion channels that shape cardiac action potentialand stabilize neuronal membrane potential. Kv7.1channel is mainly expressedin the myocardial tissue, and in association with its auxiliary subunits KCNE1channel co-encodes the slowly activating component of the delayed rectifier(IKs). The Kv7.1/KCNE1channel complex plays an important role in theregulation of action potential and mutations in both KCNQ1and KCNE1underlie a large number of hereditary arrhythmias leading to long QTsyndrome. The currents co-expressed by Kv7.2and Kv7.3have been provedto be the principal molecular basis of the M-channel. Moreover, KCNQ5canalso contribute to M-channel. Mutations in Kv7.2and Kv7.3or dysfunction ofM-channel can cause many neurologic diseases, such as benign familialneonatal convulsions (BFNC). Kv7.4mainly expressed in cochlea andvestibular organ of the inner ear and central auditory nerves. Mutations inKv7.4have been shown to cause hereditary deafness syndrome (DFNA2).Arecent study found that Kv7.1, Kv7.4and Kv7.5channel high expression invascular smooth muscle cells and multiple other smooth muscle cells.Pharmacological study showed that Kv7.1channel had no effect on vasculartension, but Kv7.4and Kv7.5channel involve in the modulation of vasculartension. In addition, the Kv7.4and Kv7.5regulators have potential value onthe treatment of hypertension. However, the high selectivity regulators ofKv7.4Kv7.5have not been reported. This research found that Fasudil, a Rhokinase inhibitor, can slectively activate Kv7.4/Kv7.5channels but have noeffect on other Kv7channels.Fasudil (also called HA1077) is a new kind of isoquinoline sulfonamidederivatives and a selective Rho kinase inhibitor, which can potently expand blood vessels and effectively alleviate cerebral vasospasm. It mainly uses forthe prevention and improvement of subarachnoid hemorrhage caused bycerebrovascular disease and the treatment of cerebral vasospasm andpulmonary hypertension in clinical. Previous studies suggested that thevasodilative effect of fasudil is closely related to the inhibition of Rho kinase.However, this research found that it is also related to the increase of Kv7current. Furthermore, we found that the modulation effect of fasudil on Kv7potassium channels is more selectively.The Kv7potassium channel families were used as a starting point in thispaper. Patch clamp technique was used to study the effect of fasudil on Kv7K+channel currents expressed in HEK293cells and M channel currentsgenerated from small rat DRG neurons. In addition, wire myograph systermwas used to study the effect of fasudil on the resistance vessels of mesentericarteries.1. The effect of fasudil on Kv7K+currents expressed in HEK293cellsObjective: To study the selective effects of fasudil on Kv7.1/KCNE1,Kv7.2, Kv7.2/Kv7.3, Kv7.4and Kv7.5channel currents.Methods: Patch clamp technique was used to study the effect of fasudilon Kv7K+currents expressed in HEK293cells. Retigabine (RTG), a Kv7potassium channel opener, was used as a positive control.Results:(1) There were no significant effects of30μM fasudil onKv7.1/KCNE1channel currents.(2)There were no significant effects of1μM,3μM,10μM,30μM and100μM fasudil on Kv7.2and Kv7.2/Kv7.3channel currents.(3) Fasudil concentration-dependently potentiated Kv7.4K+currents. Onaverage,10μM,30μM,100μM fasudil and10μM RTG increased Kv7.4K+tail currents by about0.82,1.38,2.03and3.16fold respectively.(4) Fasudil concentration-dependently potentiated Kv7.5K+currents.Fasudil make the G-V curves move left as the concentration increasing.30μMFasudil shifted G-V curves to left from-53.7mV to-62.4mV, and theamplitude was9.39±1.10mV；10μM RTG shifted G-V curves left to-68.9 mV, and the amplitude was13.925±1.80mV.Conclusions: Fasudil increase the Kv7.4and Kv7.5currents, but therewas no apparent role on Kv7.1/KCNE1, Kv7.2and Kv7.2/7.3currents. Whichindicated that fasudil selectively modulated the Kv7channel currents.2. The effect of fasudil on M currents in rat small dorsal root ganglia(DRG) neuronsObjective: To study the excitatory effects of fasudil on dorsal rootganglia (DRG) neurons generated from small rats.Methods: Patch clamp technique was used to study the effect of fasudilon Kv7/M channel currents expressed in DRG neurons that generated fromsmall rats.Results:(1) There were no significant effects of1μM,3μM,10μM,30μM and100μM fasudil on on M currents in DRG neurons generated fromsmall rats.(2) There were no significant effects of1μM,3μM,10μM,30μM and100μM fasudil on on resting membrane potential (RMP) of rat DRG neurons.Conclusions: There were no obvious effects of fasudil on M currents andRMP in DRG neurons, and it prompted that there were no obvious effects offasudil on Kv7.2/7.3currents.3. The effect of fasudil on mesenteric secondary resistance vessels isolatedfrom ratsObjective: To study the excitatory effects of fasudil on mesentericsecondary mesenteric secondary resistance vessels isolated from rats.Methods: Wire myograph systerm was used to study the effect of fasudilon mesenteric secondary resistance vessels. Each experiment was divided intotwo groups. Fasudil group: Phe (10μM) was used to pro-contract the vessels.After it smoothly, different concentrations fasudil were added into the bath inturn. And then record the result. Fasudil+XE991group: The vesselspro-contracted by Phe. After it smoothly, XE991(10μM, a specific blocker ofthe Kv7potassium channels) was added to the bath and incubation for15min.And then record the result. Results: Fasudil concentration-dependently dilated the mesentericsecondary vascular resistance pretreated with Phe with an EC50of0.83±0.25μM, and the Emax was96.12%. Fasudil concentration-dependently dilated themesenteric secondary vascular resistance pretreated with Phe+XE991with anEC50of5.04±0.96μM, and the Emax was96.46%.Conclusions: Fasudil could make dose-effect curve of the vesselspretreated by Phe+XE991to the right. The mechanisms may be that fasudilenhanced the Kv7.4and Kv7.5currents in vascular smooth muscle cells.Which indicated that the selective effects of fasudil on Kv7.4and Kv7.5is oneof the mechanisms of its vasorelaxant effectsThe above researching conclusions indicate that fasudil has selectivemodulation effects on Kv7potassium channels, and it provides a new idea forthe development of selective Kv7potassium channels. Furthermore, theeffects of fasudil on enhancing the Kv7.4and Kv7.5currents may be one ofthe mechanisms of its vasorelaxant effects, and it provides a new theoreticalbasis for the clinical application.