Rab35 GTPase Positively Regulates Endocytic Recycling Of Cardiac KATP Channels

Objective:KATP channel(ATP-sensitive K+Channel)is a kind of potassium channel located in cell membrane.KATP channel is widely distributed in different tissues and organs and expressed particularly high in myocardium,skeletal muscle,pancreas,etc.Under certain pathological conditions,the density of KATP channels on the cell membrane will decrease,such as cardiac ischemia and hyperinsulinemia.A number of studies have been shown that maintaining KATP channel surface density is key to normal insulin secretion,blood pressure and cardioprotection,and has a high therapeutic potential.KATP channel surface density is in a dynamic balance of internalization and endocytic recycling,but the molecular mechanism of KATP channel endocytic recycling is poorly understood.The Rab GTPase family in eukaryotic cells almostly regulates all kinds of membrane transport,including endocytic recycling.We aim to investigate the Rab GTPases,which are directly related to membrane protein endocytic recycling,to explore its role in KATP channel endocytic recycling and provide reference for the treatment of diseases related to KATP channel transport defects.Methods:1.Constructing plasmids of Kir6.2 and SUR2A of KATP channel subunit type in cardiomyocytes.Constructing dominant negative mutant(DN)plasmids(Rab4-S22N,Rab11a-S25N,Rab11b-S25N,Rab35-S22N)of Rab GTPase family members that are directly related to endocytic recycling.2.To determine which Rab affect the density of surface KATP channel and whether they affect the properties of KATP channel,the plasmids of the two subunits of KATP channel were co-transfected with empty vector plasmid or Rab-DN plasmids into HEK293t cells.KATP channel inside-out patch clamp,extraction of biotin-labeled surface protein and western blotting were performed.Current amplitude,single channel current and open probability of KATP channel and western blotting gray value were analyzed.3.To determine whether Rab-DN affects the surface density of KATP channel by influencing KATP channel endocytic recycling,the plasmids of the two subunits of KATP channel were co-transfected withempty vector plasmid or Rab-DN plasmids into Hela cells.Primary antibodies were incubated in cell culture to label surface KATP channels that could participate in endocytosis and endocytic recycling,and colocalization and recycling immunofluorescence were detected respectively.Colocalization analysis was performed on the images,and the size of Rab vesicle was counted.Recycling velocity was quantified.4.To explore whether PI3K-Akt signaling pathway is involved in that Rab affect the density of surface KATP channel,we constructed the constitutively active mutant(CA)plasmid of certain Rab GTPase which affects surface density of KATP channel.Rab-CA plasmids were co-transfected into HEK 293t cells with the plasmids of the two subunits of KATP channel.Patch clamp and western blotting of KATP channel were performed with LY294002,an inhibitor of PI3K-Akt signaling pathway.Current amplitude,single channel current,open probability of KATP channel and western blotting gray value were analyzed.5.To further investigate the role of Rab on KATP channel recycling in primary cardiomyocytes,the adenovirus including mCherry,Rab-DN and Rab-CA of certain Rab GTPase were generated.Rat cardiomyocyte was isolated and infected with adenovirus,and KATP channel inside-out patch clamp was performed.Current amplitude,single channel current and open probability were analyzed.Results:1.Rab35-DN reduced the density of surface KATP channel.Rab-DN were transfected into HEK293t cells.KATP channel inside-out patch clamp experiment:there was no significant difference in single channel current and channel open probability among the control group and the four Rab-DN groups,and the current amplitude of KATPchannel in Rab35-DN group was significantly smaller than that in other groups.Membrane proteins were extracted for western blotting experiment:among the control group and the four RabDN groups,surface density of KATP channels in Rab35-DN group was significantly lower than that in other groups.2.Rab35-DN decelarated the endocytic recycling of KATP channels,leading to the accumulation of KATP channels inside the cells.Rab-DN were transfected into Hela cells.Colocalization:in the four Rab-DN groups,the colocalization coefficient M2 of Rab35-DN and KATP channels was significantly higher than that of other groups,and the size of KATP channel positive vesicles in Rab35-DN was significantly bigger than that in other groups.Double staining to detect KATP channel recycling:there was no significant difference in the surface KATP channel signal between the control group and the RAB35-DN group at 0 h recycling time point,but at 2 h recycling time point,the surface KATP channel signal was significantly less in Rab35-DN group than that in control group.3.Rab35-CA up-regulated the density of surface KATP channel in a PI3K-Akt signaling independent manner.Rab35-CA was transfected into HEK293t cells and LY294002 treatment was performed.KATP channel inside-out patch clamp experiment:there was no significant difference in single channel current and channel open probability among all groups.The current amplitude in Rab35-CA group was significantly higher than that in the control group,and there was no significant difference between DMSO treatment and LY294002 treatment.Western blotting showed that LY294002 treatment significantly reduced the phosphorylation of Akt.There was no significant difference in the total expression of KATP channels between Rab35-CA and control groups,and no significant difference with or without LY294002 treatment.4.Rab35-CA increased the density of surface KATP channel in rat cardiomyocytes.Rat cardiomyocyte was isolated and infected with adenovirus.The current amplitude of KATP channel in Rab35-CA group was significantly higher than that in control and Rab35-DN groups,and there was no significant difference in single channel current and channel open probability of KATP channel among groups.Conclusions:Rab35 is a key molecule in regulating KATP channel recycling.The inactivation of Rab35 leads to the decrease of recycling rate of KATP channel,the expression density of KATP channel on the cell membrane,and the current amplitude.Activation of Rab35 can increase the current of KATP channel in cardiomyocytes,suggesting that Rab3 5 is a potential drug target for the treatment of diseases related to KATP channel transport.