Swapping of transmembrane domains in the epithelial calcium channel TRPV6

Tetrameric ion channels have either swapped or non-swapped arrangements of the S1–S4 and pore domains. Here we show that mutations in the transmembrane domain of TRPV6 can result in conversion from a domain-swapped to non-swapped fold. These results reveal structural determinants of domain swapping and raise the possibility that a single ion channel subtype can fold into either arrangement in vivo, affecting its function in normal or disease states.

We reproduced crystals of TRPV6 cryst and confirmed its non-swapped conformation. Notably, we were able to collect new crystallographic data, where both the S4-S5 and S6-TRP helix connectivity in TRPV6 cryst is unambiguously resolved in the 2Fo-Fc and Fo-Fc omit maps (Fig. 4), in stark contrast to our previous data 5 , where density for thirteen residues in S4-S5 linker (F471-Q483) was missing. Similarly, both the 2Fo-Fc and robust Fo-Fc omit maps for TRPV6* demonstrated completely different connectivity of transmembrane domains (Fig. 4) compared to TRPV6 cryst . These structural data therefore suggest that the single leucine L495 to glutamine substitution in S5 is responsible for the absence of domain swapping in TRPV6 cryst . Because the sequence of TRPV6* is closer to wild-type, the domain-swapped architecture of TRPV6 likely represents the physiologically relevant domain arrangement.
The presence or absence of transmembrane domain swapping could impact the functional behavior of TRPV6 considerably. Nonetheless, ratiometric fluorescence measurements of cells loaded with the Ca 2+ sensitive dye Fura-2 showed that the three constructs, TRPV6*, TRPV6 cryst and wild type TRPV6, demonstrate pore permeability to Ca 2+ and ion channels block by Gd 3+ (Fig. 5). Indeed, the pore architectures of TRPV6 cryst and TRPV6* are indistinguishable (Fig. 6a,b), and anomalous signals for Ca 2+ and Gd 3+ indicated that the positions of the pore cation binding sites are similar in TRPV6* (Fig. 6c-f) and TRPV6 cryst 5 . Thus, the structural and functional integrity of the TRPV6 pore is overall preserved in both domain-swapped and non-swapped folds. Despite these gross similarities, kinetic analysis indicated the rate of increase in Fura-2 signal is faster in TRPV6* compared to TRPV6 cryst (Fig. 7). Therefore, the Ca 2+ entry function is somewhat compromised in the non-swapped TRPV6 cryst construct, presumably due to differences in allosteric communication between the pore domain and the rest of the protein.   Table 1. Data collection and refinement statistics. *Highest resolution shell in parentheses. **5% of reflections were used for calculation of R free .
How does the L495Q mutation result in the non-swapped domain arrangement in TRPV6? In TRPV6*, L495 projects its side chain into a hydrophobic pocket formed by residues of S5 and S6 of one subunit and residues at the S4-S5 elbow of the adjacent subunit (Fig. 3e). By contrast, in TRPV6 cryst , Q495 is rotated by ~120° with respect to the orientation of L495 in TRPV6* and forms polar interactions with the hydrophilic pocket formed by S4-S6 and the TRP helix of the same subunit (Fig. 3f). These observations suggest that hydrophobic burial of the L495 residue plays an important role in driving the domain-swapped fold of TRPV6. Disruption of these hydrophobic interactions leads to the non-swapped fold. In other members of TRPV family, either leucine or valine occupy the position homologous to L495 in TRPV6 (Fig. 1b). Further, a hydrophobic pocket similar to TRPV6 is also present in the structures of TRPV1/2 2, 3, 6 , suggesting a conserved role for this region in determining the domain arrangement in TRPV channels.
In the case of non-swapped cation channels, the absence of domain swapping was attributed to shorter S4-S5 linkers [13][14][15][16][17] . We tested whether TRPV6 domain arrangement could be biased toward a non-swapped conformation by shortening of the S4-S5 linker, which has a well-defined helical structure in the domain-swapped TRPV6* but lacks secondary structure in TRPV6 cryst . Remarkably, TRPV6* constructs with up to ten residues deleted in the S4-S5 linker expressed robustly and could be isolated as a monodisperse species with gel filtration elution time corresponding to the tetrameric assembly (Fig. 8a,b). We determined the crystal structure of TRPV6* with four residues deleted in the S4-S5 linker (TRPV6*-del1) and confirmed its non-swapped architecture (Fig. 8c,d). Therefore, the length of the S4-S5 linker is indeed a critical determinant of domain swapping. Interestingly, ratiometric fluorescence measurements did not detect measurable Ca 2+ influx in HEK cells expressing TRPV6*-del1, suggesting that while the tetrameric expression and assembly in this mutant is preserved, gating function is significantly perturbed.
In summary, our studies demonstrate that small sequence changes can result in a major alteration of the transmembrane domain arrangement of a tetrameric ion channel. Depending on the nature of the mutation, this structural alteration can lead to strong or weak changes in ion channel function. We speculate that tetrameric ion channels might be able to switch domain arrangements under a variety of conditions, including dynamic rearrangement between distinct conformations, as a result of mutations, post-translational modifications, or other perturbations. The impact of changes in domain swapping on ion channel function may play a role in pathophysiology.
Expression and Purification. TRPV6 constructs described in this paper were expressed and purified as previously described for the TRPV6 cryst 5 . In brief, TRPV6 constructs were introduced into the pEG BacMam vector with C-terminal thrombin cleavage site (LVPRG) followed by eGFP and streptavidin affinity tag (WSHPQFEK), for the expression in suspension of baculovirus-transduced HEK293 GnTIcells. 48-72 hours after transduction with P2 virus, cells were harvested, washed with PBS at pH 8.0 and after Gd 3+ (d) calculated for wild type TRPV6 (blue), TRPV6 cryst (red) and TRPV6* (green). In (d), ΔF 340 /F 380 was measured after preincubation with Gd 3+ and subsequent addition of 2 mM Ca 2+ . sonication, cellular membranes were prepared. The protein was extracted from cellular membranes using 20 mM n-dodecyl-β-D-maltopyranoside, and purified using Strep-Tactin affinity chromatography followed by size exclusion chromatography. Crystallization and structure determination. Optimized crystals of purified TRPV6* or TRPV6*-del1 were grown in the same condition as crystals of TRPV6 cryst 5 , which included a reservoir solution consisting of 20-24% PEG 350 MME, 50 mM NaCl and 50 mM Tris-HCl pH 8.0-8.5 in hanging drop configuration at 20 °C. Prior to crystallization using hanging drop method, the protein was subjected to ultracentrifugation (Ti100 rotor, 40000 rpm, 40 min, 4 °C) to remove the aggregated protein. Crystals were cryoprotected by serial transfer into the buffer composed of 100 mM NaCl, 100 mM Tris-HCl pH 8.2, 0.5 mM DDM and 50 mM ammonium formate, containing increasing concentration of PEG 350 MME, with maximum concentration of up to 33-36%, and then flash frozen in liquid nitrogen. To obtain crystals with Ca 2+ or Gd 3+ , protein was incubated with 10 mM CaCl 2 or 1 mM GdCl 3 , respectively, for at least 1 hour at 4 °C prior to crystallization. . Arrows indicate the time at which 2 mM Ca 2+ was added. The curves were fitted with one (red) or two (blue) exponentials. Note, TRPV6* kinetics was well described by the double but not the single exponential. Over three measurements, the time constants of the single exponential fit (τ single ) were 14.5 ± 2.7 s for TRPV6* and 110 ± 16 s for TRPV6 cryst , τ fast = 2.2 ± 1.0 s, τ slow = 54 ± 11 s and A fast = 0.57 ± 0.01 for TRPV6* and τ fast = 5.4 ± 2.8 s, τ slow = 135 ± 23 s and A fast = 0.10 ± 0.03 for TRPV6 cryst . At n = 3, the values of τ single and A fast for TRPV6* and TRPV6 cryst were statistically different (t-test, P < 0.05). X-ray diffraction data collected at APS (beamlines 24-ID-C/E), NSLSII (beamline 17-ID) or ALS (beamline 5.0.2) were indexed, integrated and scaled using XDS 18 or HKL2000 19 . The initial phase information and structures were solved by molecular replacement using Phaser 20 and the structure of rat TRPV6 cryst 5 (PDB ID: 5IWK) as a search probe. Most of TRPV6*, including the ankyrin domain, S1-S4, pore module and C-terminal hook, was nearly identical to the TRPV6 cryst ; the rest of the structure was built using the omit map as a guide. The robust electron density for the S4-S5 linker was evident from the initial phases obtained by molecular replacement, and map features improved further during refinement. The model was refined by alternating cycles of building in COOT 21 and automatic refinement in Phenix 22 and Refmac 23 . All structural figures were made using the PyMOL Molecular Graphics System (Version 1.8 Schrödinger, LLC). Fura 2-AM measurements. The intracellular Ca 2+ measurements from HEK cells expressing rat TRPV6 or TRPV6 cryst or TRPV6* were performed as described previously 5 . All Fura2-AM-based fluorescence measurements were conducted using spectrofluorometer QuantaMaster TM 40 (Photon Technology International) at room temperature in a quartz cuvette under constant stirring.