The Role Of Voltage Dependent Potassium Channels In Regulationg Of Gastrointestinal Smooth Muscle Motility And Its Mechanism In Mice

Voltage dependent potassium channels (KV) are widely expressedin gastrointestinal smooth muscle and play an important role in regulationof smooth muscle motility. Based on their kinetic and pharmacologicalproperties, voltage dependent potassium currents can be divided in to twoparts: the fast inactivation component (IKVfast) and the slow inactivationcomponent (IKVslow). They play an important role in setting the restingmembrane potential and sharping the action potential. So theabnormalities of KVchannels usually cause the dysfunction of smoothmuscle motility, but there have been very few reports about it by far.Small bowel obstruction is the commonly encountered disease in surgerywhich caused by the dysfunction of the smooth muscles when thecontents pass through the small intestine. Our previous studies showedthat in partial small bowel obstruction animal model, the morphologicremodeling (hypertrophy of smooth muscle) accompanied the electricalteration and contraction abnormalities. So the partial small bowel obstruction animal model is appropriate to investigate the electricremodeling and motility dysfuction. H2S has been focused in recent yearsas an endogenous gaseous signal molecule; it has wide effects inphysiological and pathological conditions. H2S can be generated by thegastrointestinal tissue endogenously and the bacteria in the gut also candigest the content and generate plent of exogenous H2S. The smoothmuscles of gastrointestinal tract are always in the environment of higherconcentration H2S comparing with other tissues. So studing themechanism of H2S regulating gut motility has great physiological andpotential clinical significance.The molecular and electrophysiology techniques were used in thisstudy to reveal the role and mechanism of KVchannels in regulation ofsmooth muscle contraction. This study was divided into two parts. Firstly,we explored the possible relation ship between KVchannel remodelingand the dysfuction of hypertrophic smooth muscel in partial obstructionmice. We successfully isolated the single smooth muscle cells fromobstruction smooth muscle tissues, and analysed the KVcurrent density,the alteration of sensitivity to voltage and the sensitivity to KVblockers(TEA and4-AP). Western blot, immunohistochemistry andimmunoprecipitation were utilitized to detect the channel expression andphosphorylation alteration in hypertrophic smooth muscles. Secondly, weinvestigated the mechanism of exogenous H2S enhanceing gastrointestinal smooth muscle spontaneous contration. Based on theexcitatory effect of exogenous H2S on gastric antral smooth muscle withthe isometric tension measurement, we used patch-clamp and membranepotential dye to investigate the target protein of H2S. The biotin switchassay was used to test the S-sulfhydration of KV4.3Results as follows:Voltage dependent potassium channel rmodeling in murineIntestinal smooth muscle hypertrophy induced by partialobstruction：1. After14days obstruction, the gastrointestinal tract above theobstruction ring significantly dilated and the muscular layer wasobviously thickened. Intracellular recording showed that after14daysobstructon, both the amplitude and frequency of slow wave weredecreased and the resting membrane potential depoloarized in smoothmuscle, this suggests that the morphological remodeling of smoothmuscel was accompanied with electric changes in partical obstructionmouse.2. After freshly isolating the normal and obstruction small intestinesmooth muscle cells with collagenases, we found that the cells inobstruction smooth muscle hypertrophy and the membrane capacitance ofthem increased from30.32±1.84pF (n=36) of normal cells to149.37±5.47pF (n=40, p <0.05). 3. We compared the current densities of IKVamong obstruction,normal and sham groups with whole cell patch clamp. The results showedthat IKVfastand IKVslowwere increased in obstruction smooth muscles butthe current densities of IKVfastand IKVslowdecreased.4. The activation curve of IKVfastshifted to the left but the one ofIKVslowshifted to the right after14days obstruction. The V0.5actof IKVfastin normal, sham and obstruction groups are-2.78±2.64mV,-4.3±3.63mV and-15.74±3.8mV, respectively(n=12,11,13, P<0.05), while theV0.5actof IKVslowin normal, sham and obstruction groups are-8.65±2.78mV,-8.99±3.8mV and5.14±2.61mV, respectively (n=12,11,13,P<0.05). But the inactivation curves of IKVfastand IKVslowwere notaffected after obstruction. These results suggest that the sensitivity tovoltage of IKVhas been changed after obstruction.5. After14days obstruction, the IKVfastsensitivity to4-AP (KVblocker) was not affected. Compared with normal and sham groups theIC50of IKVslowto TEA was slightly decreased.15mM TEA completelyblocked the IKVslowin obstruction smooth muscle cells (93%±0.04), butin normal and sham cells it only blocked55.4±0.05%and52.7±0.05%.The results indicate that the sensitivity to TEA of IKVis increased afterobstruction. 6. Compared with normal and sham groups, the expression levels ofKV4.3and KV2.2in obstruction smooth muscle tissue were significantlyincreased. Immunohistochemistry and membrane staining double lableshowed that the KV4.3and KV2.2expression in the obstruction smoothmuscle membrane is much higher than the other two groups. Thethreonine and serine phosphorylation levels in purified KV4.3and KV2.2by immunoprecipitation of obstruction tissues were significantlyincreased compared with the other two groups.CONCLUSIONS：KVchannel remodeling happened in the partialobstruction-induced hypertrophy of smooth muscle, in which IKVcurrentdensity was decreased; IKVsensitivities to blokers and voltage werechanged. Expression of KVchannel increase and phosphorylationalteration may cause IKVcurrent density decrease leading to the restingmembrane potential depolarization in the hypertrophic smooth muscle.The excitatory effect and molecular mechanism of exogenousH2S on mouse gastric smooth muscle contraction：1. Our previous studies showed that H2S donor NaHS at lowconcentration (<200μM) have an excitatory effect on the gastric smoothmuscle contraction. To confirm this result, we firstly examine the effectof100μM NaHS on isolated gastric smooth muscles contraction. Theresults showed that NaHS significantly increased the basal tension of isolated gastric antral smooth muscle strips. KVchannel blocker4-AP (5mM) blocked the excitatory effect of NaHS.2. In the presence of strong reducer DTT (200μM), the excitatoryeffect of NaHS on basal tension was diminished. Alternatively, theexcitatory effect was reversed after administration of DTT. But200μMDTT alone can not affect gastric smooth muscle spontaneous contraction.3. As4-AP blocked the excitatory effect of NaHS on gastric smoothmuscle contraction, we utilized whole-cell patch clamp to test the effectof NaHS on the IKVin isolated gastric smooth muscel cells.100μMNaHS inhibited the IKVfastrather than IKVslowin isolated antral smoothmuscle cells. Thiol group of protein blocker NEM and DTT blocked theinhibitory effect on IKVfast.4. To further determine the target of NaHS, we heterologouslyexpressed KV4.1, KV4.2and KV4.3in H293cells and observed the effectsof NaHS on them.500μM NaHS inhibited the KV4.3current induced bystep depolarization but did not affect KV4.1and KV4.2currents. Theinhibitory effect could be blocked by NEM but not MTSET.5. Since NaHS inhibited the IKVresulted in depolarization ofmemebrane potential, we utilized the membrane potential dye DiBAC4(3)to test the membrane potential of cultured gastric antral smooth musclecells and observed the effect of NaHS on the membrane potential.200μM NaHS induced depolarization of smooth muscle cells, which could be inhibited by Pretreatment with NEM. Knock down of KV4.3with siRNAalso inhibited the depolarization.6. Biotin switch assay was used to test the S-sulfhydration of KV4.3.500μM NaHS sulfhydrated KV4.3expressed in H293cells, which couldbe inhibited by NEM pretreatment.100μM NaHS sulfhydrated KV4.3ingastric smooth muscle tissues, which could be reversed by200μM DTT.CONCLUSIONS：①Exogenous H2S enhances gastric smooth muscletension through S-sulfhydration of KV4.3resulting in the inhibition of thevoltage-dependent potassium current in mice.②KV4.3plays animportant role in regulating membrane potential of gastrointestinalsmooth muscle which may be the target of exogenous biochemicalmaterials in regulating gastrointestinal smooth muscle motility.SUMMARYThe above results can be summarized as follows:1. KV4.3subunit in KVchannels of gastric smooth muscle plays animportant role in regulation of smooth muscle membrane potential.2. KV4.3may be the target of exogenous and endogenous factors inregulating smooth muscle membrane potential. 3. H2S inhibited the IKVin smooth muscle through Sulfhydration ofKV4.3leading to the depolarization of membrane potential to display theexcitatory effect on smooth muscle.