Interactions Between Heterotrimeric G Proteins And S-type Anion Channels And Their Effects On Channel Activity

Heterotrimeric G protein(G protein for short)is an important and conserved signal transduction molecule that mediates signal transmembrane between membrane extracellular and effectors on the intracellular,and is composed of three subunits:a,?and y.In Arabidopsis there is a typical G protein a subunit GPA1,a typical ? subunit AGB1,two typical ? subunits AGG1 and AGG2,and an atypical G protein y subunit AGG3.Studies in animal cells have shown that an important downstream target protein directly regulated by G protein is ion channels.The analysis of G protein subunit loss-of-function mutants in plants shows that G protein is involved in the signal transduction of stomatal movement regulated by a variety of stimuli and is related to its influence on the activity of anion channels.However,it is still unclear whether plant G proteins interact with anion channels and whether the interaction directly affects channel activity.In this paper,three kinds of protein interactions research methods and electrophysiological techniques are mainly used to study the physical interaction of each subunit of G protein with S-type anion channels SLAC1 and SLAH3 and the effect of G protein on the activity of SLAC1 channel,and analyzed the role of G protein in ABA-induced stomata closure,the main experimental results and conclusions obtained are as follows:1.The results of the yeast two-hybrid membrane protein showed that GPA1,AGG1,AGG2 and AGG3 can interact with ion channels SLAC1 and SLAH3,while AGB1 cannot interact with SLAC1 and SLAH3;the ion channels are divided into N-terminal,transmembrane region and C-terminal,the analysis results of the yeast two-hybrid system using membrane protein and nucleoprotein f1urther showed that the key regions for GPA1 to interact with SLAC1 and SLAH3 are all transmembrane regions,and the key regions for AGG1,AGG2,and AGG3 to interact with SLAC1 and SLAH3 are all C-terminal and N-terminal.2.The results of bimolecular fluorescence complementation technology showed that G protein subunits GPA1,AGB1,AGG1,AGG2 and AGG3 can interact with ion channels SLAC1 and SLAH3 in the cell membrane area of tobacco epidermal cells.3.The pull-down experimental results further verified that GPA1 can directly interact with the transmembrane regions of the two ion channels SLAC1 and SLAH3 in vitro;AGB1 can directly interact with the N-terminus of SLAC1 and SLAH3 in vitro.However,it does not directly interact with the C-terminus of the two ion channels;AGG1,AGG2,and AGG3 can all directly interact with the N-terminus and C-terminus of SLAC1 and SLAH3 in vitro.4.SALC1,different subunits of G protein and OST1 or Ca2+dependent protein kinase CPK21 heterologous expression in Xenopus laevis oocytes,using two-electrode voltage clamp technology to detect the activity of SLAC1 channel,the experimental results of show that:different subunits of G protein,G?y dimers and G protein trimers can not directly activate the channel activity of SLAC1,and have a certain inhibitory effect on the basic activity of SLAC1;OST1 or CPK21 can activate the channel activity of SLAC1,but the subunits of G protein,G?y dimer and G protein trimer can all inhibit the activation of SLAC1 channel activity by OST1 or CPK21 to varying degrees,their inhibitory effects are as follows:the descending order is:G protein trimer>GPydimer>AGGs>AGBl>GPAl.5.The voltage clamp results showed that the inhibitory effect of G protein y subunit AGG1,AGG2 or AGG3 on the activation of SLAC1 channel activity by OST1 was dose-dependent.The pull-down technique was used to study the effect of different doses of AGG3 on the interaction between OST1 and SLAC1.The results showed that as the amount of AGG3 increased,the amount of OSTl that interacted with SLAC1 continued to decrease,suggesting that AGGs may inhibit the activation of SLAC1 channel activity by OST1 by inhibiting the interaction between OST1 and SLAC1.Considering that the key regions where AGGs and OST1 interact with SLAC1 respectively have the intracellular N-terminal,it is speculated that AGGs and OST1 may inhibit the interaction of OST1 and SLAC1 by competing for binding sites.Secondly,after co-expression of two continuous kinase-independent SLAC1 channels SLAC1F450A and SLAClT513D with AGG3,the voltage clamp results show that AGG3 can inhibit the channel activity of SLAC1T513D to a certain extent,but cannot inhibit the channel activity of SLAC1F450A.It suggests that the interaction between AGGs and intracellular of SLAC1 may also directly inhibit the channel activity of SLAC1.6.Comparing the time course of stomata closure of Arabidopsis wild-type and G protein mutants gpa1-3,agb1-1,agg1-lc,agg2-l and agg3-1 under ABA treatment,the results showed that in gpa1-3,agb1-1 and agg3-1 mutants,the degree of stomata closure in the early stage of ABA treatment(less than 1 h)was significantly greater than that of wild-type and aggl-1c and agg2-1 mutants,while during ABA treatment,the degree of stomata closure of agg1-1c and a1gg2-1-1c was not significantly different from that of the wild type,and after 1 h of ABA treatment,there was no significant difference between the degree of stomata closure of Arabidopsis wild-type and the above-mentioned G protein mutants.The results suggest that G protein subunits GPA1,AGB1 and AGG3 are all involved in the early stages of ABA-induced stomatal closure as negative regulators.This result combined with the above-mentioned protein interaction and voltage clamp results and previous research results suggest that the signal transduction pathway of ABA-induced stomatal closure may be:in the absence of ABA,heterotrimeric G protein binds to anion channels such as SLAC1 to inhibit the basic activity of the channel and the binding of other activating factors to the channel,so that the anion channel is completely closed,so the stomata are opened;when ABA exists,on the one hand,ABA promotes the activation of protein kinases such as OST1 through its signal transduction pathway;on the other hand,ABA promotes the dissociation of heterotrimeric G protein into free Ga and G?? dimers,and at the same time,it is possible to reduce the expression of G protein by inhibiting the expression of G protein gene,which not only reduces the amount of G protein bound to the anion channel,but also promotes activated protein kinases such as OST1 to bind the anion channel SLAC1 and SLAH3 and activate the anion channels by phosphorylation which in turn causes the stomata to close.