A channelopathy mechanism revealed by direct calmodulin activation of TrpV4

Abstract

Ca 2+ -calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca 2+ increases TrpV4 activity through Ca 2+ -CaM binding to a C-terminal Ca 2+ -CaM binding domain (CBD). We confirmed this model by directly presenting Ca 2+ -CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca 2+ -CaM. Deleting a 10-residue upstream peptide (Δ795-804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca 2+ -CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca 2+ -CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.skeletal dysplasia | calcium | ion channel | autoinhibition | TrpV1

A competing hydrophobic tug on L596 to the membrane core unlatches S4–S5 linker elbow from TRP helix and allows TRPV4 channel to open

“…Massive injection of cRNA, however, could often compromise the health of the oocytes, causing larger leak current and complicating interpretation. We have therefore contrived a L596A E797K double mutant harboring a second mutation in the cytoplasmic domain that boosts the basal Po (17). Though the E797K channel had current activation and inactivation kinetics typical of GOF mutants (17), the double mutant (standard 5-ng injection) again only showed the decaying outward current even upon strong depolarizations (Fig.…”

“…S3 E and F); this could be because they are not sensitive to swelling or because their very low Po obscured even a several-fold increase. We therefore tested the L596A E797K double mutant using second mutation to magnify the basal Po (see above) (17). The L596A E797K double mutant was responsive to cell swelling (Fig.…”

“…Topology and sequence conservation indicate important functions of the S1-S4 peripheral domain and the S4-S5 linker in TRPV channels, likely similar to those in K v (7,8). The TRP helix is followed by cytoplasmic domains used in Ca 2+ -calmodulin regulation (17); it also has bonds with the pre-S1 helix and the preceding domains at the N-terminal end, and is therefore capable of receiving cytoplasmic stimuli. The TRP helix-S4 linker elbow contact might unlatch in response to the motion of the ankyrin repeats that have been implicated in gating (10) and inactivation (24).…”

Transient receptor potential vanilloid 4 (TRPV4) activation by arachidonic acid requires protein kinase A–mediated phosphorylation

“…The alpha-helix with Ser-824 also resides in the previously reported calmodulin (CaM)-binding domain (CBD), which is thought to be involved in Ca 2ϩ -dependent regulation of TRPV4 (61)(62)(63). Interestingly, TRPV4 mutants with deleted CBD (63) or charge-reversal (5R/E) within CBD (61) show increased channel activity in two different expression systems.…”

When size matters: transient receptor potential vanilloid 4 channel as a volume‐sensor rather than an osmo‐sensor

“…TRPV4 thus appears to act as a polymodal sensory protein and is reported to be critical for regulation of intracellular Ca 2+ signalling, osmo-and mechano-transduction and temperature-sensing, as well as cell volume regulation and the maintenance of Ca 2+ homeostasis (Ye et al 2012;Jo et al 2015;White et al 2016). Hereditary TRPV4 channelopathy mutations lead to a variety of phenotypic disorders (Dai et al 2010;Nilius & Voets, 2013;Loukin et al 2015) and the expression pattern and molecular characteristics of TRPV4 point to its putative involvement in pathophysiological conditions with increased pressure and edema formation, in skeletal dysplasias, inflammatory responses, and sensory/motor neuropathies (Ye et al 2012;Jo et al 2015;Ryskamp et al 2015). Mice with genetic deletion of TRPV4 display impaired regulation of systemic tonicity (Liedtke & Friedman, 2003), although the molecular links between (i) altered osmolarity of the extracellular fluid and TRPV4 activation and (ii) TRPV4 activation and cell volume regulation remain elusive (Liedtke & Friedman, 2003;Nilius et al 2004;Becker et al 2005;Liedtke, 2005b;White et al 2016).…”