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. 2018 Aug 15;4(8):eaau6088.
doi: 10.1126/sciadv.aau6088. eCollection 2018 Aug.

Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6

Appu K Singh  1 , Luke L McGoldrick  1   2 , Edward C Twomey  1   2 , Alexander I Sobolevsky  1
Affiliations

Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6

Appu K Singh et al. Sci Adv. .

Abstract

Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)-mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore's intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design.

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Figures

Fig. 1
Fig. 1. Structure of the hTRPV6-CaM complex.
(A to D) Side (A and B), top (C), and bottom (D) views of hTRPV6-CaM with hTRPV6 subunits (A to D) colored cyan, green, yellow, and pink, and CaM colored purple. Calcium ions are shown as green spheres. In (B), only two of four hTRPV6 subunits are shown, with the front and back subunits removed for clarity. Side chains of TRPV6 residue W583 and CaM residue K115 are shown as sticks. (E) Expanded view of CaM bound to the proximal and distal portions of the TRPV6 C terminus. Side chains of CaM residues coordinating calcium ions in the EF hand motifs and K115 are shown as sticks.
Fig. 2
Fig. 2. Contact interfaces between CaM and TRPV6.
(A) Surface representation of rTRPV6-CaM viewed intracellularly with TRPV6 subunits and CaM colored similarly to Fig. 1. Boxes indicate rTRPV6-CaM interfaces expanded in (B) to (G). (B to G) Interfaces between CaM and different regions of rTRPV6, with residues involved in rTRPV6-CaM binding shown as sticks and calcium ions as green spheres.
Fig. 3
Fig. 3. Inactivated TRPV6 pore.
(A) Ion conduction pathway (cyan) in hTRPV6-CaM, with residues lining the selectivity filter and around the gate shown as sticks. Only two of four hTRPV6 subunits are shown, with the front and back subunits removed for clarity. (B) Pore radius calculated using HOLE (41) for hTRPV6-CaM (inactivated state, green), hTRPV6 (PDB ID: 6BO8; open state, orange), and hTRPV6-R470E (PDB ID: 6BOA; closed state, cyan) (13). Dashed line corresponds to 1.4 Å (radius of a water molecule). (C and D) S6 helices in superposed structures of the inactivated (green), open (orange), and closed (cyan) states described in (A), with the residues contributing to gating shown as sticks. The portion of CaM encompassing K115 is shown in (C).
Fig. 4
Fig. 4. Mechanism of TRPV6 inactivation.
Cartoons represent the structural changes associated with CaM-induced inactivation of TRPV6. Only two of four TRPV6 subunits are shown, with the front and back subunits removed for clarity. The transition from the open to inactivated state involves tilting of the lower portions of the S6 helices toward the center of the pore at the alanine A566 gating hinge and closure of the pore stopping the permeation of ions (green spheres). The loss of the R589-Q473 salt bridges, which stabilize the α-to-π helical transition in S6 in the open state, is compensated by a cation-π interaction between K115 and the π-system of four tryptophans forming a cage at the pore intracellular entrance.
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