The Functional Role Of N-termini Of TRPCs In Homo-and Heteromerization And In Ca²⁺ Entry

As Ca²⁺-nonselective cation channels，transient receptor potential canonical （TRPC）channels exist extensively in nonexcitable and excitable cells. TRPC consists of sevenmembers including TRPC1, C2, C3, C4, C5, C6, C7, which can be categorized into fourgroups: TRPC1, TRPC2, TRPC3/6/7and TRPC4/5. In human, TRPC2is a pseudogeneand cannot be expressed. The other6members can be divided into two functionalsubgroup: one is TRPC1, C4, C5and the other is TRPC3, C6, C7. Functional TRPCchannels depend on four subunits of TRPC molecules to connect with each other andform a tetrameric ion channels. Based on sequence comparison and structuralpredictions, the structure of TRPC channels is similar to that of voltage-gated cationchannels, which have4subunits. This kind of structure model was verified in3Dstructures of TRPC3using low-resolution （15–35） electron cryomicroscopy （cryo-EM）.All of the TRPCs can not only form multimers with themselves as homomeric structures,but also connect with each other between different TRPCs as heteromeric structures. Forheteromultimers, there are different types:（1） initially, it was verified that thecombination of different TRPCs can only occurred within one functional subgroup, suchas between TRPC1, C4, C5, or between TRPC3, C6, C7, members from differentsubgroup cannot associated with each other, like TRPC4cannot connect with TRPC3;（2） later, some research focused on TRPC1, which discovered that TRPC1was a linker molecular between two functional subgroup, like TRPC1can connect with TRPC4andTRPC6;（3） then, more and more research showed that any two kinds of TRPCs canform heteromultimers, no matter they are members of one functional subgroup or fromdifferent functional subgroup. Now, it is generally considered that any one of the TRPCscan form homomultimers and any two of them can form heteromultimers, ignoring theirgroups. But it is still illusive that how this association happens. The exact region ofTRPCs responsible for the connection is still unknown. Till now, only seldom studyrevealed that the self-association of TRPC4was depended on the N-termimus of TRPC4.For other members, there is no study focusing on the region responsible for connection.Those methods used before were often CoIP, GST-pull down, circular dichroism （CD）,which cannot present the protein distribution and physiological conditions in livingcells.On the other hand, it is hot point of study on the functions of TRPC channel inrecent years. There were plenty of research indicating that as calcium channel, TRPCsparticipate in receptor-operated calcium entry （ROCE） and store-operated calcium entry（SOCE）. It is well-known that TRPC3, C6, C7display Gd3+-resistant-ROCE, and anykind of TRPCs show phenomenon of Tg-induce-SOCE. Otherwise, the exact functionaldomain of TRPCs which is responsible for their function on calcium entry is larglyunknown.The purpose of the present study was to determine the association of TRPCs by anew method bimolecular fuorescence complementation （BiFC）, which can observe thefluorescence signals due to protein interactions in living cells. We cotransfected twointact TRPC-VN155/VC155, or one intact TRPC-VN155/VC155and one N-mutationalTRPCΔN-VC155/VN155in HEK293, and then check the fluorescence intensitybetween two expressing system and find out their difference, thus verify the role ofN-termini of TRPCs in their connection. As a supplemental approach, CoIP and WesternBlot were also used to further prove the importance of N-termini. In addition, we usedCa²⁺image assay to determin the effect of Gd3+on ROCE of intact TRPC3, C6, C7or N-mutational TRPC3ΔN, TRPC6ΔN, TRPC7ΔN, and the effect of Tg on SOCE ofintact TRPC1, C4, C5or N-mutational TRPC1ΔN, TRPC4ΔN, TRPC5ΔN, in order toconfirm the importance of N-termini of TRPCs in calcium entry through TRPCchannels.BiFC assays showed that cells expressing intact TRPC-VC155and TRPC-VN155formed a circle around the cell membrane, while the signals in cells expressing samekind TRPC-VC155and TRPCΔN-VN155were obviously attenuated. CoIP and WesternBlot showed that any kind of TRPCs-VN155were immunoprecipitated by the intactTRPC-VC155, but not by the same kind N-mutational TRPCΔN-VC155. Thisphenomenon not only occurred in same TRPCs systems, but also in variant TRPCsystems. Ca²⁺image showed that intact TRPC3, C6, C7-VN155/VC155manifestedGd3+-resistant-ROCE, while the phenomenon vanished for the N-mutational TRPC3ΔN,TRPC6ΔN, TRPC7ΔN-VN155/VC155. Intact TRPC1, C4, C5-VC155exhibitedTg-induced SOCE, while the phenomenon disappeared for N-mutational TRPC1ΔN,TRPC4ΔN, TRPC5ΔN-VC155.To sum up, this research further confirmed that any kind of TRPCs could formhomomultimers, and any two of them could form heteromultimers. Whatever how theyassociated, the N-terminus was essential for this connection. What’s more, theN-terminus was also essential for their calcium permeability of TRPC channels. It isthus clear that the N-termini of TRPCs exert key functional role in homo-andheteromultimers of TRPCs and in calcium entry through TRPC channels.