The Regulation And Mechanism Of KV2.1 On The Secretory Function Of Islet ? Cells Through The Interaction Of Endoplasmic Reticulum And Cell Membrane

In eukaryotic cells,Endoplasmic Reticulum(ER),as the organelle with the largest intracellular membrane area,can form a special area in close contact with different membrane organelles-Membrane Contact Sites(MCSs).The MCSs formed between the ER and the plasma membrane(PM)rely on the tethering effect of the protein to maintain a small distance in the dynamic or transient range of 7-30 nm,and have a role in intracellular Ca²⁺signaling and lipid transport important meaning.The repolarization of?-cell action potential is mediated by delayed rectified potassium current.Voltage-gated potassium channel(Kv)subtype Kv2.1 is the main component of delayed rectified potassium current and is expressed at high levels in?-cells.Earlier studies found that the expression of?-cell KV2.1 channels is very abundant,however,the use of channel-specific inhibitors to inhibit the activity of these channels(including the related KV2.2)has little effect on the action potential of?-cells and has no effect on the secretory activity of insulin.However,several research groups reported that the use of KV2.1 knockout mice revealed that the knockout of the Kv2.1channel protein changed the glucose-induced electrical activity of pancreatic beta cells and promoted insulin secretion.In short,the role of Kv2.1 and KV2.2 channels in insulin secretion is still contradictory and controversial,and it is necessary to further explore the correlation and molecular mechanism of Kv2 channels and?-cell secretion function.Recent studies have shown that the Kv2.1 channel exists at the ER-PM junction and participates in the formation of neuronal ER-PM contact points.It is a new ER-PM contact point link protein that forms a cluster structure on the cell membrane.However,this kind of physiological functions remain unclear.This study confirmed that the KV2.1 channel regulates?-cell secretory activity,which is the regulatory structure that promotes the formation of ER-PM MCSs.The direct interaction between KV2.1channel and VAPA protein was observed,and the correlation between this ER-PM interaction relationship and?-cell secretory activity was analyzed.1.Kv2.1 channel regulates islet?cell secretory activity In order to confirm the regulation effect of KV2.1 channel on insulin secretion of?-cells,the effects of silencing KV2.1 protein gene and restricting current conduction on insulin exocytosis of?-cell line INS-1 cells were examined.The results of patch-clamp membrane capacitance measurement showed that Si RNA knockdown of Kv2.1 channel protein expression in INS-1 cells can significantly inhibit vesicle secretion,while Kv2.1 channel-specific blocker STX(stromatoxin)can significantly inhibit the outward potassium current is interrupted,but it has no effect on vesicle secretory activity.The above results confirm that KV2.1 channel has an effect on vesicle secretion when it is not conductive.2.Formation of Kv2.1 channel at the contact point of ER-PM in?cell To confirm the multi-aggregation phenomenon of KV2.1 channels on?-cell PM.EGFP fluorescently labeled KV2.1 overexpressing INS-1 cells,using confocal laser scanning imaging technology,scanning imaging at the bottom and middle of the cell showed that KV2.1 was expressed on PM of INS-1 cells and formed a patchy aggregate structure distribution.To further verify the effect of KV2.1 channel on insulin secretion under high Ca²⁺conditions.Kv2.1-EGFP and with m Cherry-STIM1 co-transfected INS-1 cells,a control group and TG group.TG(Thapsigargin)is a Ca²⁺-ATPase inhibitor,which can release the intracellular Ca²⁺pool by opening the calcium channel of the cell membrane,thereby increasing the intracellular Ca²⁺concentration.Using confocal laser scanning imaging technology,imaging at the bottom of the cell showed that,compared with the control group,the TG group obviously induced the migration of STIM1 to the KV2.1-mediated ER-PM contact position,and highly co-localized with KV2.1.In previous studies,STIM1 was found to be a regulatory protein secreted by?cells.The above experimental results suggest that the KV2.1 channel and STIM1 may interact with each other at the ER-PM contact site to regulate the secretion function of?cells.3.The interaction of Kv2.1 channel and VAPA mediates the formation of?-cell ER-PM contact point In order to study the interaction between KV2.1 channel and VAPA,mediate ER-PM membrane contact.KV2.1protein was fluorescently labeled with EGFP,and VAPA protein was labeled with m Cherry.In endogenous HEK293 cells were over-expressed EGFP-KV2.1 and co-expressed EGFP-KV2.1+m Cherry-VAPA.Using confocal scanning to scan the bottom of the cell shows:when only the KV2.1plasmid is transferred,it is evenly distributed on the PM and does not form a cluster-like typical structure;when co-transferred into KV2.1 and VAPA,they are highly co-localized and form a cluster-like distribution on the membrane.Endogenous HEK293 cells do not express VAPs protein.In order to further confirm the molecular mechanism of the interaction between KV2.1 and VAPA,EGFP-Kv2.1 and VAPA base point mutant m Cherry-VAPA(K94D/M96D)were used to co-transform INS-1 cells.Confocal scanning of the bottom of the cell revealed that it significantly inhibited the formation of cluster-like structures mediated by the Kv2.1 channel.The above experimental data proves that KV2.1 can establish correct contact with VAPA to mediate ER-PM contact.4.FRET experiment confirms that Kv2.1 channel directly interacts with VAPA In order to verify the direct interaction between KV2.1 and VAPA,KV2.1 and VAPA were labeled with fluorescent Clover2 and m Ruby2,and Clover2-N1+m Ruby2-VAPA and Clover2-KV2.1+m Ruby2-VAPA were converted into INS-1cell,respectively.Clover2 and m Ruby2 are excited with 488nm and 559nm respectively,and the fluorescence intensity of the donor fluorescent group Clover2 and the acceptor fluorescent group m Ruby2 are recorded at 500-530nm and 570-625nm,respectively.Confocal FRET imaging technology was used to obtain fluorescence images of donor channel,FRET channel,and acceptor channel,and Nfret values were calculated:it showed that Clover2-Kv2.1 and m Ruby2-VAPA had obvious FRET phenomenon,and Clover2-N1 and m Ruby2-VAPA had no obvious FRET phenomenon.These results suggest that the Kv2.1 channel and the VAPA protein coexist in the same ER-PM contact point,and the direct link maintains the formation of the ER-PM contact point.5.ER-PM contact point mediated by Kv2.1 channel promotes?-cell secretion function To investigate whether ER-PM mediated by KV2.1channel affects?-cell secretion,WT-INS-1 cells were used as a control group,Clover2-Kv2.1 and m Ruby2-VAPA were co-transformed into INS-1 cells as a positive group,Clover2-Kv2.1 and m Ruby2-VAPA(K94D/M96D)were co-transformed into INS-1 cells as a negative group.All groups were treated with high glucose(20 m M)for 1 hour.The detection of insulin secretion by radioimmunoassay showed that the positive group was significantly higher than the control group,and the negative group was significantly lower than the control group.These experimental data show that KV2.1 can promote the exocytosis of?cells when contacting with VAPA to construct ER-PM;loss of VAPA mediated reduction of insulin secretion.Conclusion:KV2.1 channel is an important regulator of the secretory function of pancreatic islet?cells.Its direct interaction with VAPA protein maintains the formation of ER-PM contact sites,which is closely related to the molecular mechanism of KV2.1 channel regulating islet?cell secretion function.