Forward Genetic Analysis Reveals Multiple Gating Mechanisms of TRPV4

Abstract

TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4␣PDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca 2؉ block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate. Transient receptor potential (TRP)2 channels are a functionally and evolutionarily diverse group of cation-selective channels characterized by polymodal gating to chemical, thermal, mechanical as well as other stimuli (1). Predicted transmembrane topology and cryomicroscopic images (2) indicate that TRPs are structurally similar to other cationic channels such as the voltage-gated K ϩ channels (3). Each of the four subunits (4) is modeled to have six transmembrane helices (S1-S6), where S1-S4 forms the peripheral domain and the four S5-S6 domains converge to form the core, with the S6 lining the ion pathway. An ion filter is found in the pathway near the outer half (5). Structural homology to K ϩ channels (3) indicates (6, 7) and direct mutational analysis supports (8) that the cytoplasmic ends of the four S6 conditionally converge to occlude the pathway, creating an intracellular gate.TRPV4, of the vanilloid class of TRP channels, originally drew attention and was isolated based on its ability to respond to hypotonic stress (9, 10). It was subsequently found to also be activated by heat (11), the arachidonic acid (AA) metabolite 5Ј,6Ј-epoxyeicosatrienoic acid (5Ј,6Ј-EET) (12) as well as other compounds (13,14), but the most potent activator was found to be an exogenous agonist, 4␣-phorbol 12,13-didecanoate (4␣PDD) (15). 4␣PDD and hypotonicity were found to activate TRPV4 through distinct pathways (16). In cultured kidney cells, hypotonicity is thought to activate TRPV4 through the production of 5Ј,6Ј-EET, possibly by activating phospholipase A2 (16). There must be other mechanisms by which TRPV4 is activated by hypotonicity, since it maintains robust hypotonic activation when expressed in yeast, which is incapable of synthesizing polyunsaturated fatty acids such as AA and its metabolites (17). TRPV4 is expressed in a broad range of tissue types that have varying physiological relevance (18). TRPV4Ϫ/Ϫ knock-out mice are compromised...

Wild-type and Brachyolmia-causing Mutant TRPV4 Channels Respond Directly to Stretch Force

“…Recent forward genetic screening for TRPV4 mutations that are toxic to yeast yielded GOF alleles nearby (L619P, L624P). These yeast-selected mutations have similar but much stronger GOF phenotypes than R616Q (23). These findings support a general Shaker-like topology for TRPV4, with S5 being part of the main gate.…”

“…Xenopus oocytes 3 days after injection of 4 ng of wild-type TRPV4 cRNA presented small currents that could be greatly amplified by the addition of the synthetic phorbol ester activator 4␣-PDD at 3 M (n Ͼ 30) (Fig. 1a, upper) (23). As expected of TRPV4, these currents show prominent outward rectification (24).…”

“…Oocytes rarely survived injection of Ͼ40 ng of wild-type TRPV4 cRNA or Ͼ4 ng of R616Q cRNA. The toxicity of stronger TRPV4 GOF mutants selected through yeast genetics has also been observed (23).…”

“…In previous work, the apparent specificity of BPB inhibition of the hypotonic response could have reflected a partial inhibition of PLA 2 due to the shorter incubation, resulting in obvious inhibition of only the weaker hypotonic response. It is notable that depolarization activation was not substantially affected by BPB in the absence of additional stimulation, underscoring the independence of voltage-from chemical-and mechanical-dependent gating (23). Anthranilic acid, another PLA 2 inhibitor, at 20 M for 15 min (27) also had no effect on the TRPV4 hypotonic response.…”

Yeast Luminometric and <em>Xenopus</em> Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

“…Because yeast makes no PUFAs such as AA or EET, and its genome has no PLA2 or epoxygenase genes, this expression also shows that they are not required for TRPV4 to sense force. Having TRPV4 in the molecular biologically most amenable eukaryotes also allows efficient forward-or reverse-genetic manipulations 25 .…”