2.1 The Non-covalent Interactions Form Multiple Hairpin Topological Loops along the Gating Pathway of Oxidized mTRPV3 in the Closed State at 42⁰C
mTRPV3 has a high temperature coefficient (Q10=27) . It is mainly expressed in skin keratinocytes and oral and nasal epithelia, mediating thermal reception and pain sensation [5-6]. Like other TRPV channels, TRPV3 is a homotetramer. Each monomer has S1-S6 as transmembrane domain (TMD) and a large intracellular amino- (N-) terminal as ankyrin repeat domain (ARD). S1-S4 form a voltage-sensor-like domain (VSLD) while S5-S6 and the pore helix and two pore loops are folded as a pore domain. Both the VSLD and the pore domain are swapped via an S4−S5 linker. The TRP helices, which are almost parallel to the membrane, interact with both the skirt ARD and the TMD. Several lipid sites were also found in their interfaces. The pre-S1 domain, together with the carboxy- (C-) terminal loop domain, couples TMD with ARD. The residues 638GLGD641 in the P-loop-extended region line the selectivity filter to permeate partially hydrated Na+ , K+ or Ca2+ ions but not to function as a upper gate. In contrast, the narrowest pore constriction around M677 on S6 may act as a lower gate [18, 21].
C612 and C619 in the outer pore of mTRPV3 can form a disulfide bond upon oxidization . In the closed state at 42⁰C, the diversity of non-covalent interactions between amino acid side chains in oxidized mTRPV3 can produce multiple hairpin topological loops along the gating pathway from D396 in the pre-S1 domain to K505 in the TRP domain. First, salt bridges are present between several charged pairs. For example, R416 on pre-S1 and D519 in the S2-S3 linker, K432 on pre-S1 and E704 in the TRP domain or D396 on pre-S1, D586 and K589 on S5, E631 and K634 on the pore helix, E682 on S6 and K686 in the TRP domain, E687 and K690, E689 and R693, R698 and E702 in the TRP domain (Figure 1).
Second, p-p interactions appear between several pairing aromatic residues. They include: F441 on S1 and W433 on pre-S1 or Y565 on S4, F445 and F449 on S1 or Y565 on S4, Y448 on S1 and F526 on S3 or Y565 on S4, W493 and F489 on S2 or F447 or Y451 on S1, F526 on S3 and Y564 on S4, Y540 on S3 and Y547 on S4, Y564 and Y565 on S4, F590 and Y594 on S5, Y622 in the first pore loop and F654 on S6. In addition, R696 in the TRP domain forms a cation-p interaction with W433 on pre-S1 while F590 on S5 and L673 on S6 form a CH-p interaction (Figure 1).
Third, H-bonds emerge between different hydrophilic residues. These H-bonding pairs are T397 and K432 on pre-S1 or E704 in the TRP domain, Q529 on S3 and Y448 or Y451 on S1, T566 on S4 and S576 in the S4-S5 linker, R567 on S4 and T699 in the TRP domain, Q570 in the S4-S5 linker and E689 in the TRP domain, D586 on S5 and T680 on S6, Y594 on S5 and Y661 on S6, E679 and N683 on S6. In addition, the vanilloid site phosphatidylcholine (PC) also relates W521 on S3 to Q580 in the S4-S5 linker via H-bonds. Of special note, there are several H-bonding pairs such as E610-K614, S624-D627, S621-Q646, T636-Y594/Y661, N647-E610/K614 in the pore domain (Figure 1).
Taken together, one largest hairpin with a 23-residue loop is present to control the D519-R416 salt bridge in the VSLD/pre-S1 interface. It starts with D519 to R416, W433, F441, Y565, F445, F449, Y448 and ends with F526. The total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 are 44 and 117, respectively (Figure 1).
2.2. The Largest Hairpin Melts at 42⁰C for Oxidized mTRPV3 Activation with an Obvious Decrease in the Total Hairpin Sizes along the Gating Pathway
In the heat-activated sensitized state at 42⁰C, oxidized mTRPV3 has several changes. In the pore domain, the N647-E610/K614, S621-Q646, S624-D627 and E679-N683 H-bonds are broken while the S620-Q646 H-bond emerges. In the S4-S5 linker/TRP interface, the Q570-E689 H-bond is disconnected. In the TRP domain and its interfaces with the pore domain and the pre-S1 domain, the E682-K686 and E689-R693 salt bridges are replaced with the N683-K686 and E689-R693 H-bonds, respectively. In addition, the T397-K432/E704 H-bonds are disrupted while the E405-K705 salt bridge and the E423-T427 and E418-R690 H-bonds are present. In the VSLD and its interface with the pre-S1 domain, the p-p interactions shift from F445-Y565 and W493-Y451 to F526-Y565 and F489-F450, respectively, with the broken W493-F489 p-p interaction. The D519-R416 salt bridge is substituted by the S511-N412 and R509-N410 H-bonds. Of special interest, in the VSLD/S4-S5 linker interface, the T566-S576 H-bond is broken. However, D519 forms a salt bridge with R567 when a vanilloid site PC in the closed state disappears  (Figure 2).
Together, after the largest hairpin with a 23-residue loop in the VSLD/pre-S1 interface melts, two largest hairpins follow. One has a 9-residue loop from S620 to Y622, F654, Q646 and back to S620 in the pore domain. The other contains an 8-residue loop from D519 to F526, Y565, R567 and back to D519 in the VSLD/S4-S5 linker interface. The total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 become 41 and 87, respectively (Figure 2).
2.3. Two Smaller Hairpins in the S4-S5 Linker/VSLD Interface Are Required to Open Oxidized mTRPV3
When oxidized mTRPV3 is finally open from the sensitized state, there are small changes in the outer pore loop. The E610-K614 H-bond is replaced with the K616-D618 salt bridge. The D586-K589 salt bridge is disconnected while the S624-D627 H-bond is restored. In the meanwhile, a CH-p interaction is present between Q646 and F654, producing the smallest hairpin with a 1-residue loop from S620 to Y622 and F654 and Q646. Thus, the largest hairpin appears in the pore domain with a 10-residue loop from D586 to F590 and L673 and T580 and back to D586 (Figure 3).
In stark contrast, in the TRP domain and its interface with the pre-S1 domain, the E687-R690 and E405-K705 salt bridges and the T397-E704 H-bond are broken. In the meantime, the E689-R693 H-bond becomes a salt bridge and T411 forms a new H-bond with R416. In the VSLD/pre-S1 interface, the N410-R509 and N412-S511 H-bonds are replaced with the T411-S515 and N412-Q514 ones. In the VSLD, the p-p interaction shifts from F450-F489 to Y451-W493. The H477-Y540 H-bond and the H471-Y547 p-p interaction also produce a new small hairpin with a 5-residue loop. When the T566-S576 H-bond is reinstated in the S4-S5 linker/VSLD interface, the resultant second largest hairpin with a 9-residue loop, together with an 8-residue loop from D519 to F526 and Y564 and Y565 and R567 and then back to D519, may be required for mTRPV3 opening. By all accounts, in the open state, the total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 become 41 and 71, respectively (Figure 3).
2.4. Reduced mTRPV3 Has the Different Largest Hairpin in the Outer Pore
In the closed state without the C612-C619 disulfide bond, reduced mTRPV3 is different than oxidized one. In the pore domain, the D586-T680, E610-K614, S621-Q646, S624-D627 and N647-E610/K614 H-bonds are disrupted together with the F590-L673 and Y622-F654 CH/p-p interactions and the E631-K634 salt bridge. In the meanwhile, the S613-N647 and E631-N643 H-bonds and the F625-V629 and N647-F654 CH/lone pair-p interactions are formed. By all accounts, the largest hairpin with a 17-residue loop in the pore domain is born from S613 to F625, V629, E631, N643, N647, and then back to S613 (Figure 4).
However, the largest hairpin in the VSLD/pre-S1 interface, which is present in the oxidized mTRPV3 (Figure 1), is unavailable because the D519-R416 salt bridge has been replaced with the new Q514-T411/N412 H-bonds to produce the smallest hairpin with no residue in the loop. When the Y451-W493 p-p interaction is disconnected with the formation of the K500-E702 salt bridge, a smaller hairpin with a 9-residue loop is present from W493 to K500, E702, R698, R696, W433, F441, Y565, Y448, F447 and then back to W493. In the S4-S5 linker/TRP interface, the R567-T699 H-bond is substituted by the R567-Q695 H-bond. In the S4-S5 linker/pore domain interface, the D586-T680 H-bond is replaced with the K581-E687 salt bridge. In the TRP domain and its interface with the pre-S1 domain, the E687-R690 salt bridge is broken but the R693-H426 and Q695-K705 H-bonds are generated. In the closed state, reduced mTRPV3’s total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 are 40 and 98, respectively (Figure 4).
2.5 Reduced mTRPV3-Y564A Has the Largest Hairpin in the VSLD/Pre-S1 Interface
When reduced mTRPV3 has a mutation Y564A in the VSLD, the channel is closed at 4⁰C. However, the disruption of the Y564-Y565/F626 p-p interactions induces several changes along the gating pathway from D396 to K705 . In the VSLD, the p-p interactions rearrange from Y565-F445 and Y540-Y547 and W521-F524 pairs to F542-Y544 and F450-F489 ones. In the VSLD/pre-S1 interface, the Q514-T411/N412 H-bonds are replaced with the D519-R416 salt bridge, and the H-bond shifts from T397-K432/E704 to T397-S402. In the VSLD/TRP interface, the K500-E702 salt bridge is disconnected. In the TRP/pre-S1 interface, the R696-W433 cation-p interaction and the H426-R693 H-bond are disrupted. In the TRP domain, the Q695-K705 H-bond is broken while the E687-R190 salt bridge and the R696-W692 CH-p interactions are present. In the S4-S5 linker/TRP interface, when the K581-E687 salt bridge is broken, the H-bond shifts from Q570-E689 to Q570-R693. In the TRP/pore domain interface, the E682-K686 salt bridge is broken. In the pore domain, the D586-T680 H-bond and the F590-L673 CH-p interaction appear between S5 and S6 while the S613-N647 H-bond is replaced with the Y622-S626 one. As a result, the largest hairpin shifts from the 17-residue loop in the pore domain to a 23-residue loop in the VSLD/pre-S1 interface, which is the same as shown in the closed and oxidized mTRPV3 (Figures 1 and 5A).
2.6 Reduced mTRPV3-Y564A opens at 37⁰C with the Melted Largest Hairpin and a Small Change in the Total Hairpin Sizes along the Gating Pathway
The Y564A mutant opens at 37⁰C with a low temperature coefficient (Q10=1.21) . When the largest hairpin melts at 37⁰C, several changes occur with the mutant. In the VSLD, the p-p interactions shift from W433-F441 and F450-F489 and F526-Y565/Y448 pairs to Y448-Y451 and H477-W481 ones. In the VSLD-pre-S1 interface, the D519-R416 salt bridge is replaced with the T411-S515 H-bond. In the VSLD-TRP interface, D519 forms a salt bridge with R698. In the TRP/pre-S1 interface, the K432-D396/E704 and R698-E702 salt bridges and the T397-S402 H-bond are replaced with the W433-R696-W692 cation-p interactions and the H417-E689 H-bond and the E702-K705 salt bridge. In the S4-S5 linker/TRP interface, the Q570-R693 H-bond is replaced with the Q570-W692 CH-p interaction. In the TRP/pore domain interface, the E689-R693 salt bridge and the E679-N683 H-bond are disrupted. In the pore domain, the CH/lone pair/p-p interactions emerge between F597-F601/L664 and F601/Y661 and F565-T660 pairs in the S5-S6 interface. However, the Y622-S626 and E631-N643 H-bonds are broken with the formation of the E631-K634 salt bridge. In this case, two larger hairpins, which link the VSLD and the TRP domain and the pre-S1 domain together, may be required for reduced mTRPV3-Y564A opening. One has a 16-residue loop from W493 to K500, E702, Q695, R567, Y565, Y448, Y449 and back to W693. The other has a 14-residue loop from S515 to D519, R698, R696, W692, E689, H417, T411 and back to S515. In any way, the total numbers of all non-covalent interactions and hairpin sizes along the gating pathway from D396 to K705 have small changes from 36 and 101 to 34 and 95, respectively (Figure 5).