| Potassium ion(K+)channels control the transport of potassium ions into and out of cells.Potassium ion channels are divided into class of voltage-activated potassium channels(Kv),inward rectifier potassium channels(Kir),calcium-activated potassium channels(KCa),and two-hole potassium channels(K2p).The voltage-gated potassium channel has 78 members,and that is a large family of approximately 40 genes.It divided into 12 subfamilies(Kv 1-Kv 12).The Kv 7 family,also known as the KCNQ family,contains five alpha subunits,Kv 7.1-7.5 that encoded by the gene KCNQ 1-5 is an attractive drug target.The Nav?subunit(Nav?1-Nav?4)is a multifunctional type I transmembrane protein that regulates the gating and localization of Nav?subunits.The Nav?1 subunit also regulates the function of some Kv channels,but its subtype and molecular regulation mechanism are still unclear,and there are rarely reports on the regulation of KCNQ channel function by Nav?1 subunit.Extracellular matrix(ECM)is a three-dimensional macromolecular network,and composed of structural proteins,fibronectin and some proteoglycans.Tenascins is a group of extracellular matrix glycoproteins composed of six members,such as Tenascin-C(TNC)and Tenascin-R(TNR).Among them,TNC and TNR are involved in cell proliferation,migration,axonal pathology,myelination,and synapses plasticity.KCNQ channels and extracellular matrix(TNC,TNR)have important physiological effects for cell proliferation,differentiation,and survival.Then,whether the KCNQ potassium channel function on the cell membrane is affected by extracellular matrix TNC/TNR? There are few reports on the regulation of potassium channels by ECM.The aim of this study is to determine the molecular interaction and regulation mechanism of KCNQ potassium channel,h Nav?1subunit and extracellular matrix(TNC/TNR).It will provide a new strategies for the prevention of clinical related diseases and new KCNQ channel targeted therapy.Herein,we specifically transfects KCNQ 1,KCNQ 2,KCNQ 3,and KCNQ 4 into HEK 293 T.And co-transfects KCNQ 1+h Nav?1,KCNQ 2+h Nav?1,KCNQ 3+h Nav?1,KCNQ 4+h Nav?1 and incubation of Tenacins(TNC/TNR),in-depth study of KCNQ+h Nav?1,KCNQ-TNC/TNR,KCNQ+h Nav?1-TNC/TNR current.These results will explore the KCNQ potassium channel,h Nav?1 subunit and extracellular matrix(TNC/TNR)molecular interaction combined regulation mechanism.1.Gated dynamics of the KCNQ channelIn the whole cell patch clamp mode,the KCNQ 1,KCNQ 2,KCNQ 3,and KCNQ 4were respectively transfected into HEK 293 T cells and the currents were recorded.The peak current,I-V curve,and Id curve were obtained.The results showed that the peak current of KCNQ 1 was about 400±50 p A,and the current density was about 70±5 p A/p F.KCNQ 2 peak current is about 350±50 p A,and the current density is about 20±5 p A/p F.KCNQ 3 has a peak electricity of about 600±50 p A and a current density of about 20±5p A/p F.KCNQ 4 peak current is about 550±50 p A and current density is about 15±5p A/p F.2.Molecular interaction between KCNQ channel and h Nav?1 subunitIn whole-cell patch clamp mode,the KCNQ 1,KCNQ 2,KCNQ 3,KCNQ 4 and h Nav?1 subunit were separately co-transfected into HEK 293 T cells and the currents were recorded.The peak current,I-V curve,and Id curve were obtained.The results show that h Nav?1 enhances the peak current of KCNQ 1 and has almost no effect on the peak currents of KCNQ 2,KCNQ 3 and KCNQ 4.Nav?1 reduced the current density of KCNQ 1,significantly increasing the current density of KCNQ 3 and KCNQ 4,but had no effect on the current density of KCNQ 2.The results showed that the h Nav?1 subunit combined with the KCNQ potassium channel could differentially regulate the gating characteristics of the KCNQ potassium channel under certain conditions.3.Modulation of KCNQ channels by extracellular matrix(Tenascin C/Tenascin R)In whole-cell patch clamp mode,the KCNQ 1,KCNQ 2,KCNQ 3,and KCNQ 4were respectively transfected into HEK 293 T cells added to the extracellularmatrix(TNC/TNR),and the currents were recorded.The peak current,I-V curve,and Id curve were obtained.Experimental results: TNC enhanced the peak current of KCNQ 2,and suppressed the peak current of KCNQ 3 and KCNQ 4,but had no effect on the peak current of KCNQ 1.TNR significantly increased the peak current of KCNQ 1,KCNQ 2and KCNQ 4,minus The peak current of KCNQ 3 is reduced.The TNC reduces the current density of KCNQ 1 and KCNQ 3 and enhances the current density of KCNQ 2and KCNQ 4.TNR reduces the current density of KCNQ 1 and KCNQ 3,increases the current density of KCNQ 2,and the KCNQ 4 The current density has almost no effect.The results showed that extracellular matrix(TNC/TNR)can differentially regulate the electrophysiological properties of KCNQ potassium channels.4.Regulation of extracellular matrix(TNC/TNR)on the combination of KCNQ channel and Nav?1 subunitIn whole-cell patch clamp mode,the KCNQ 1,KCNQ 2,KCNQ 3,KCNQ 4 and h Nav?1 subunit were separately co-transfected into HEK 293 T cells cells added to the extracellular matrix(TNC/TNR),and the currents were recorded.The peak current,I-V curve,and Id curve were obtained.Results: TNC significantly increased the peak currents of KCNQ 1+h Nav?1,KCNQ 2+h Nav?1,KCNQ 4+h Nav?1,and had no significant effect on the peak current of KCNQ 3+h Nav?1.TNR significantly inhibited KCNQ 1+h Nav?1,KCNQ 3+h Nav+h Nav?1,The peak current of h Nav?1 enhances the peak current of KCNQ 2+h Nav?1 and has almost no effect on the peak current of KCNQ4+h Nav?1.TNC has almost no effect on the current density of KCNQ 1+h Nav?1,increasing the current density of KCNQ 2+h Nav?1 and decreasing the current density of KCNQ 3+h Nav?1 and KCNQ 4+h Nav?1.TNR significantly increases KCNQ 2+h Nav?1.Current density of KCNQ 4+h Nav?1,reduced KCNQ 1+h Nav?1,KCNQ 3+h Nav?1current density.The results showed that extracellular matrix(TNC/TNR)interacted with KCNQ potassium channel and h Nav?1 subunit to affect the gating characteristics of potassium channel.Conclusion: h Nav?1 and TNC/TNR can differentially regulate the currents ofKCNQ 1,KCNQ 2,KCNQ 3 and KCNQ 4.TNC/TNR can change the current of KCNQ1+h Nav?1,KCNQ 2+h Nav?1,KCNQ 3+h Nav?1,KCNQ 4+h Nav?1,and activate specificity.Therefore,the molecular interaction control mechanism between KCNQ,h Nav?1,and Tenascins can helps to provide new ideas for the analysis of differential functional expression of potassium channels and targeted drug screening. |
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