| Objective: Kv7(Kv7.1–Kv7.5),a subfamily of voltage-gated K+channels encoded by the KCNQ genes,play an important role in regulation of neuronal excitability(Kv7.2-Kv7.5)and the cardiac action potential(Kv7.1).It is a common consensus that Kv7.2 and Kv7.3,existing in a heterotetramer are the principal molecular components of the slow voltage-gated M-channel,which widely regulates neuronal excitability.Recently,Kv7.4 and Kv7.5 have also been identified in multiple vascular smooth muscle cells and implicated to play a crucial role in regulation of vascular smooth muscle contractility and vascular tone.Kv7 channel modulators provoke significant changes in vascular smooth muscle membrane potential and vascular tone.In vascular smooth muscle cells,the physiologically predominant molecular architecture is a Kv7.4/Kv7.5 heterotetramer.Thus there seems a distinguished expression of neuronal(Kv7.2/Kv7.3)and vascular(Kv7.4/Kv7.5)Kv7 channels which provide a selective drug action strategy.Non-selective Kv7 modulators may have side effect liabilities in the clinic as such the first approved Kv7 opener retigabine for treatment of epilepsy(Potiga(Ezogabine))has been warned for the limited use due to the side effect,e.g.urinary retention(the bladder smooth muscle cells are also the target of Kv7 modulators including the openers.Most of the reported Kv7 modulators show poor selectivity across these five subtypes,especially among Kv7.2-Kv7.4.For example,retigabine and flupirtine have been shown to activate all neuronal Kv7 channels(that is Kv7.2–7.5)although does not affect Kv7.1.Thus,compounds that are selective across Kv7 subtypes,at least act selectively on either neuronal or vascular Kv7 channels,have a significant advantage in therapeutic potential.Fasudil,the sole clinically available RhoA/Rho kinase(ROCK)inhibitor,is already in use to treat cerebral vasospasm,as well as to improve the cognitive decline seen in stroke victims,and its anti-angina effect has also been confirmed in humans.In an earlier preliminary study,we found fasudil selectively activates Kv7.4/Kv7.5 but did not affect Kv7.2/Kv7.3.In the present study,we are going to investigate the underlying molecular mechanism.Methods: Patch clamp technique,homology modeling and molecular docking simulation technique,and directed point mutation technique was used to study the molecular basis of fasudil on Kv7.4 K+ currents.Results:1 The selective effect of fasudil on Kv7 K+ currents expressed in HEK293 cells.Fasudil(30 μM)slightly inhibited Kv7.1/KCNE1 currents,did not affect Kv7.2,Kv7.2/7.3 currents,enhanced Kv7.4 and Kv7.4/Kv7.5 currents expressed in HEK293 cells.2 Homology modeling and molecular docking result for the binding of fasudil within the pore domain of Kv7.4(KCNQ4)channel.Homology Modeling and Molecular docking simulation results indicates existing of interaction between fasudil and Ile308 in S6 of the Kv7.4 channel.3 Construction of Kv7.4(S6)mutantsKv7.4(S6)mutants: Kv7.4(I308V),Kv7.4(L306I),Kv7.4(L306I/I308V),Kv7.4(G302T/A304T)were constructed by directed point mutation technique.4 The effect of fasudil on the Kv7.4(S6)point mutants by patch clamp technique.Fasudil(30 μM)potentiated the Kv7.4(G302T/A304T)currents to a similar extent as it did for the currents of Kv7.4 wildtype,but the potentiation effect of RTG(10 μM)on these mutant channels were greatly reduced.On the other hand,the responses of Kv7.4(I308V),Kv7.4(L306I)and Kv7.4(L306I/I308V)mutant channels to fasudil were greatly attenuated whereas the responses of these mutant channels to retigabine were maintained.These data indicate that the Ile308 and Leu306 in S6 of Kv7.4 are critical forthe selective potentiation effect of fasudil.5 Construction of Kv7.4(S5-Pore)mutantsKv7.4(S5-Pore)mutants: Kv7.4(W242L),Kv7.4(V248C),Kv7.4(F251L),Kv7.4(D262G/A263E),Kv7.4(T278L)were constructed by directed point mutation technique.Conclusions: Our data indicate that the Ile308 and Leu306 in S6 of Kv7.4 are critical for the selective potentiation effect of fasudil. |
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