Structural dynamics at cytosolic interprotomer interfaces control gating of a mammalian TRPM5 channel

Abstract

The transient receptor potential melastatin (TRPM) tetrameric cation channels are involved in a wide range of biological functions, from temperature sensing and taste transduction to regulation of cardiac function, inflammatory pain, and insulin secretion. The structurally conserved TRPM cytoplasmic domains make up >70 % of the total protein. To investigate the mechanism by which the TRPM cytoplasmic domains contribute to gating, we employed electrophysiology and cryo-EM to study TRPM5-a channel that primarily relies on activation via intracellular Ca²⁺. Here, we show that activation of mammalian TRPM5 channels is strongly altered by Ca²⁺-dependent desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca²⁺ binding, whereby formation and dissolution of cytoplasmic interprotomer interfaces appear to control activation and desensitization of the channel. This study shows the importance of the cytoplasmic assembly in TRPM5 channel function and sets the stage for future investigations of other members of the TRPM family.

Keywords: TRP channels; cryo-EM; electrophysiology; ion channels; molecular mechanism.

Fig. 1.

Mammalian and zebrafish TRPM5 channels possess a distinct activation profile. (A–E) Representative current time courses elicited by pulses from 0 to ±140 mV on inside-out patches obtained from transiently transfected HEK293 cells exposed to intracellular solutions containing free Ca²⁺ concentrations denoted by the horizontal bars. Dotted lines denote the zero-current level. (F) Mean ± SEM currents obtained from experiments as in (A–E), normalized to the peak current at +140 mV in the presence of 5.4 mM free Ca²⁺. Data from individual experiments are shown as circles (n = 5 to 12). Statistical significance was determined with a Tukey HSD test. Statistically significant differences are denoted by the number of asterisks according to the P-values provided in Dataset S1. Nonstatistically significant differences are denoted as n.s. (G) Current magnitudes at +140 mV from individual patches obtained from cells transfected with GFP only, or GFP together with rat (black) or zebrafish (blue) TRPM5 channel cDNA [as in (A), (C) and (E)]. For each patch, data are shown in control solution and in the presence of 25 μM (steady state) or 5.4 mM (peak) free Ca²⁺. Source data included in Dataset S1.

Fig. 2.

Structure of the rTRPM5 in the presence of EGTA. (A) Structure of the rat TRPM5 channel in the presence of EGTA is asymmetric. Individual protomers are overlaid to show that the asymmetry is most pronounced in the cytoplasmic domains, and especially in the MHR1/2 domain (blue cartoon) and the coiled coil (CC, red cartoon). (B) Each cytoplasmic interprotomer interface is unique in rTRPM5_EGTA. The MHR domains of each protomer are shaded differently (A: red; B: marine; C: purple; D: gold). The MHR domains of protomer A (red) extend deep into the cytosol so that the dimensions of the channel from the top of the transmembrane domain (S764) to the bottom of the MHR domain (W296) are ~135 Å. The MHR domains in protomers B (purple) and D (gold) appear to rotate upward, which shortens the length of the channel to ~125 Å as well as the distance to the neighboring protomers. (C) Close-up of the boxed region in (B). The conformation of the MHR domains also dictates the distance between protomers. In protomer A (red), where the MHR domains are extended into the cytosol, the distance to the neighboring protomer D is large (23 Å between the Cα of K94 of MHR A and N1090 of MHR D). This distance shortens upon rotation of the MHR domains, as observed in MHR B, C, and D.

Fig. 3.

The coiled-coil–MHR1/2 dynamics do not significantly affect activation and desensitization of rTRPM5 by Ca²⁺. (A) Bottom–up view of the rat TRPM5 channel determined in EGTA. Four distinct interfaces are observed between the MHR1/2 domain and the CC of neighboring protomers in this structure. They are colored in red (interface I, MHR A and CC B), blue (interface II, MHR B and CC C), purple (interface III, MHR C and CC D), and gold (interface IV, MHR D and CC A). (B) The distinct interfaces are caused by the arrangement of the CC. The proximal part adopts a tetrameric coiled-coil conformation, but the distal part is highly asymmetric. The density for the CC assembly is rendered as mesh and shown at contour level 0.15. (C) An overlay of the individual CC helices shows that the asymmetry begins at the flexible GG hinge. (D) Representative time courses at ±140 mV obtained from inside-out patches from cells expressing rTRPM5 channel mutants G1092A, G1093A, G1092A G1093A, and the deletion construct Δdistal CC. The dotted line denotes the zero-current level. (E) Group data for experiments in (D), shown as mean ± SEM (n = 5) or as individual experiments (open circles). Data were normalized to the peak current at +140 mV and 5.4 mM free Ca²⁺. Statistical significance was determined with a Tukey HSD test. Statistically significant differences are denoted by the number of asterisks according to the P-values provided in Dataset S4. Nonstatistically significant differences are denoted as n.s. Source data for (C and D) included in Dataset S4.

Fig. 4.

Trace amounts of calcium induce large conformational changes in rTRPM5. (A) Comparison of rTRPM5_EGTA with the conformations of rTRPM5 determined in the presence of trace amounts (~0.7 μM) of Ca²⁺ (rTRPM5_trace-1, lilac; rTRPM5_trace-2, purple; rTRPM5_trace-3, blue). The Top panel shows a side view. The cytoplasmic domains are highlighted. The dashed line represents the distance between MHR1/2 helix Cα K94 and the rib helix Cα N1090. The length of the channel (the distance between the Cα carbons of the S2 residue S764 and the MHR1/2 residue W296) condenses from 135 Å in rTRPM5_trace-1 to 125 Å in rTRPM5_trace-3. The Bottom panel shows a top view. The shortening of the channel coincides with a widening of the TM domains, as measured from the Cα of S2 residues V768 of opposing protomers. (B) Alignment of rTRPM5_trace-1, rTRPM5_trace-2, and rTRPM5_trace-3 tetramers_. For clarity, only a single protomer is shown for each structure. The alignment reveals a rotation around the rib helix (indicated by the box and enlarged Inset). The rib helix of rTRPM5_trace-3 is rotated by 12° and displaced laterally by ~4 Å compared to rTRPM5_trace-1. The red dot signifies the position of P1045 in the rib helix. (C) An overlay of the individual protomers shown in (B). (D) A schematic describing the conformational changes observed in rTRPM5_trace.

Fig. 5.

Structures of rTRPM5 in the presence of high Ca²⁺. (A) A side-by-side comparison of rTRPM5_high1-4 structures: rTRPM5_high-1, light orange; rTRPM5_high-2, gold; rTRPM5_high-3, deep orange; rTRPM5_high-4, maroon. The Top panel shows a side view, and the cytoplasmic domains are highlighted. The dashed line indicates the distance between MHR1/2 α2 helix Cα K94 and the rib helix Cα N1090 of neighboring protomers. The length of the channel is measured between the Cα of the S2 residue S764 and the MHR1/2 residue W296. The Bottom panel shows a top view. (B) An overlay of individual protomers of rTRPM5_trace-3 (blue) and rTRPM5_high-4 (maroon)—structures which exhibit the highest degree of coupling in the two datasets. Notable differences include a small offset in the position of the CC (shown in the red box) and the conformation of the pore (dashed line box). (C) A close-up of the interface between S4, S4–S5 linker, S5, and S6 in rTRPM5_trace-3 (blue) and rTRPM5_high-4 (maroon).

Fig. 6.

The interprotomer contacts are functionally important. Interfaces between MHR1/2 and MHR3 and the rib helix of neighboring protomers in rTRPM5_EGTA (A and B) and rTRPM5_high-4 (C and D). (A) Side view of the interface between MHR1/2 and MHR3 and the rib helix from the neighboring protomer. Only the MHR1/2 α2 helix is shown for clarity. The distance between the Cα atoms of K94 and N1060 measures ~23 Å. (B) Top view of the interface in rTRPM5_EGTA including the α3 helix of the MHR1/2 domain. (C) Side view of the MHR1/2 and MHR3 and the rib helix interface in rTRPM5_high-4. The distance between the Cα atoms of K94 and N1060 measures ~12 Å. (D) The top view of the interface shows how α2 and α3 from MHR1/2 and HTH α4 of the neighboring MHR3 condense around the rib helix. The blue residue labels in (C) and (D) reflect results described in (E). (E) Mean ± SEM inside-out patch currents from cells expressing WT or mutant rat TRPM5 channels as shown on SI Appendix, Fig. S15. Data were measured at ±140 mV, normalized to peak at +140 mV and 5.4 mM free Ca²⁺, and quantified at each of the indicated intracellular free Ca²⁺ conditions. Data from individual experiments are shown as circles (n = 4 to 8). Statistical significance was determined with a Tukey HSD test. Statistically significant differences are denoted by the number of asterisks according to the P-values provided in Dataset S6. Nonstatistically significant differences are denoted as n.s. Bars are colored black for WT and WT-like mutants and blue for mutants with significantly reduced responses to 25 μM free Ca²⁺. (F) Mean ± SEM inside-out patch currents from cells expressing WT- or mutant zebrafish TRPM5 channels as in SI Appendix, Fig. S15 and depicted as in (E) (n = 5 to 6). Source data for (E and F) included in Dataset S6.

Fig. 7.

The role of cytoplasmic domains in mammalian TRPM function. (A) A cartoon representation of the proposed rTRPM5 mechanism. In the absence of Ca²⁺, the cytoplasmic domains possess a high degree of flexibility and assume a range of conformations. However, the cytoplasmic interprotomer interfaces remain uncoupled. The channels can open when Ca²⁺ is bound and the cytoplasmic interprotomer interfaces are fully coupled. Finally, during desensitization, a relaxation occurs—the coupling between protomers decreases and the open state becomes destabilized. Conformational changes remain concerted between all four subunits in the presence of Ca²⁺. (B) Cytoplasmic interfaces in some existing TRPM structures. Cytoplasmic domains have been shaded for clarity.