Structural insights into the TRPV4-RhoA complex offer clues to solve the puzzle of TRPV4 channelopathies

The transient receptor potential vanilloid type 4 (TRPV4), which belongs to the TRP (transient receptor potential) ion channel family, is expressed in various cell types throughout the body. After being activated by endogenous ligands and various exogenous stimuli, such as mechanical forces, warm temperatures, osmotic pressure, and various chemicals, TRPV4 functions as a nonselective cation channel that facilitates the transport of calcium, sodium, and other cations across the cell membrane. TRPV4 is an essential component of numerous cellular responses and physiological effects [1].

TRPV4 inhibition is emerging as an intriguing therapeutic approach because genetic mutations related to the gain-of-function of the TRPV4 channel have been linked to multiple diseases and ailments, which present a variety of clinical symptoms [2]. RhoA is a small GTPase protein that functions as a molecular switch, alternating between an active, GTP-bound state and an inactive, GDP-bound state, in order to dynamically regulate cellular processes. Recent studies have identified RhoA as a direct interactor of TRPV4, which suppresses TRPV4 ion channel function. Disruption of the TRPV4-RhoA interaction may contribute to the pathological effects of neuropathy-causing TRPV4 mutations [3, 4]. Although the initial Cryo-EM and X-ray structures of Xenopus tropicalis TRPV4 provide insights into the ion permeation and gating mechanisms of TRPV4 [5], the functional and structural characterization of the human TRPV4-RhoA complex is still largely unknown.

The cryo-EM three-dimensional structures of full-length wild-type human TRPV4 and its complex with the GTPase RhoA were reported in two recent publications published on June 23, 2023, by the Lee group [5] and the Sobolevsky group [6]. A two-layered tetramer composed of four identical subunits comprises the reported human TRPV4 structures. While the transmembrane region (top layer) is composed of the voltage-sensor-like domain (VSLD) and a central ion channel pore, the intracellular region (bottom layer) consists of the cytosolic N-terminal ankyrin repeat domain (ARD), where the RhoA molecules bind. There is also a coupling domain (CD) that bridges the VSLD and ARD. These findings have advanced our understanding of the interaction between ion channels and small GTPases and have immediate practical implications for the development of therapies targeting TRPV4.

The two research teams have shed important light on the mechanisms governing TRPV4 channel gating and inhibition by examining the structural mechanisms of the channel in both the antagonist-bound closed state and the agonist-bound open state. The Sobolevsky group discovered the binding sites for the TRPV4 agonist 4-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle. Meanwhile, the Lee group demonstrated that the TRPV4 agonist GSK101 and antagonist GSK279 could have opposite effects on TRPV4 gating, depending on the ligand-dependent coupling or decoupling of the VSLD-TRP helix-CD subdomains. Interestingly, both GSK101 and GSK279 interact with the same cavity in the TRPV4 protein.

It should be mentioned that the two studies have reported different findings regarding the pore conformation dynamics in the TRPV4 structures when exposed to several TRPV4 agonists/antagonists. The Sobolevsky group demonstrated that the pore domain in the selectivity filter (I678-G679-M680) and the gate (from M718 to I715) became wider in the agonist 4-PDD-bound open state compared to the inhibitor HC-067047-bound closed state. However, the Lee group discovered that the gate (from M718 to I715) in the agonist 4-PDD-bound open state is wider, while the selectivity filter (I678-G679-M680) is narrower compared to the inhibitor GSK279-bound closed state. The cryo-EM structures show that GSK279 is located in a cavity between the TRP domain and the VSLD of TRPV4 and it is stabilized by several aromatic and polar residues within the VSLD cavity. The GSK279 binding region does not overlap with TRPV4 disease mutations. GSK279 is notably clinically safe and orally bioavailable. The unique structural properties of TRPV4 in complex with GSK279 shed light on the mechanism underlying ligand-dependent TRPV4 gating and may assist in the development of TRPV4-specific therapies. Furthermore, a recent study has also discovered that ruthenium red, a commonly used inhibitor for many TRP channels, directly blocks the entrance of the pore and narrows the selectivity filter to inhibit the mammalian TRPV4 channel [6]. These findings suggest that various agonists/antagonists may induce specific alternations in the pore conformation of TRPV4 structures. This should be considered in future studies that examine the impact of other TRPV4 agonists/antagonists on the conformation of the channel pore.

When a TRPV4 antagonist is present, RhoA molecules attach to the intracellular ARD of hTRPV4 and prevent it from moving, thereby stabilizing TRPV4 in the closed state. In contrast, when a TRPV4 agonist is present, the activation of TRPV4 leads to calcium influx, which in turn releases RhoA from ARD and causes ARD to move towards the membrane. This transformation results in TRPV4 transitioning from a closed state to an open state. Perturbing the interface between TRPV4 and RhoA by introducing gain-of-function mutations into either TRPV4 or RhoA increases the activity of the TRPV4 channel. This emphasizes the significance of RhoA as an auxiliary subunit for TRPV4 in the regulation of TRPV4 channel activity and intracellular calcium homeostasis. The significance of this connection in TRPV4-related disorders is also suggested by disease-associated mutations at the TRPV4-RhoA interface. By focusing on the refinement of the RhoA and ARD region of the TRPV4 structure, the Lee group made an additional discovery. They found that the mutations in TRPV4 that cause neuropathy are located at the interface between TRPV4 and RhoA, and they disrupt the interactions between the two proteins. Additionally, a number of RhoA mutations linked to cancer also disrupt the TRPV4-RhoA binding interface, supporting a lack of RhoA-mediated TRPV4 inhibition in TRPV4-linked diseases. These structural studies have revealed previously unknown mechanisms that underlie neuropathy caused by TRPV4 mutations. They have also provided valuable insights into the development of potential structure-based therapeutics to alleviate or prevent TRPV4 mutation-related neuropathy.

Fig.1.

Cryo-EM structures of the human TRPV4-RhoA complex in the antagonist-bound closed state, agonist-bound open state, and in the presence of neuropathy-causing mutations. Created with BioRender.com.