Mutant TRPV4-mediated Toxicity Is Linked to Increased Constitutive Function in Axonal Neuropathies

Abstract

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.Scapuloperoneal spinal muscle atrophy (SPSMA), congenital distal spinal muscle atrophy (CDSMA), and Charcot-MarieTooth disease type 2C (CMT2C, also known as hereditary motor and sensory neuropathy type 2, HMSN IIC) are phenotypically heterogeneous and dominantly inherited disorders involving topographically distinct muscles and nerves (1, 2). Genetic linkage analysis mapped the disease loci to an overlapping region at 12q24 (3-5). Recently, it has been shown that these three axonal neuropathies are allelic disorders caused by mutations in the gene encoding the transient receptor potential cation channel, subfamily V (vanilloid), member 4 (TRPV4) 3 (6 -8). To date, seven mutations in TRPV4 have been identified in 15 families with axonal neuropathies (6 -10). Functional studies, however, yielded divergent hypotheses about the pathogenic mechanism of the diseases (6 -8, 10).In our original study, we tested two mutations, R316C and R269H, which were identified in the original SPSMA and CMT2C families, respectively (6). We found that in HEK293 cells, mutant TRPV4 had a physiological localization on the plasma membrane similar to wild-type TRPV4 (wtTRPV4). Calcium imaging showed that mutant TRPV4 had significantly increased calcium channel activity, at both basal and activated levels when compared with wtTRPV4. These findings were consistent with electrophysiological recordings, which showed a higher TRPV4 conductance in HEK293 cells expressing the R269H mutant compared with wtTRPV4. In another study, Landouré et al. (8) showed similar results in HEK293 cells and Xenopus laevis oocytes with R269H and R269C mutations. Furthermore, mutant TRPV4-mediated cytotoxicity was observed in transfected HEK293 cells and dorsal root ganglion neurons. This cytotoxicity was related to increased intracellular calcium concentrations (8). More recently, we have also shown that two other TRPV4 mutants (R232C and R316H) localize appropriately and cause reversible and toxic hypercalcemia in HEK293 and HeLa c...

TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P2

“…Intracellular Ca 2 þ signals are crucial for diverse cellular processes such as survival and death; therefore, cells expressing the TRPV4 mutants might struggle to maintain [Ca 2 þ ] i at non-toxic levels. Indeed, propidium iodide uptake experiments revealed that the expression of the N296D-H299P mutant, as well as the R269H and R315W mutants 9,10 , caused a marked increase in cell death (Fig. 8c).…”

“…The mutations in TRPV4 are the most recently identified CMT subtype, in terms of genetic locus 1,2 . Previous studies have suggested that the gain-of-function mutations of TRPV4 in these diseases, which exhibit increased basal and maximum Ca 2 þ channel activities, are clustered within the TRPV4 ARD and affect arginine residues, especially at the R232C, R269C/H, R315W, R316C/H positions [7][8][9][10] . The increased Ca 2 þ influx and subsequent Ca 2 þ overload associated with these mutations are thought to be the cause of cell death, and have been proposed as a mechanism for peripheral axonal degeneration in TRPV4-dependent neuropathies 9,10 .…”

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

“…The calculation was performed using a T of 3 ms (Fecto et al, 2011), V of 10 fl, a volume similar to that occupied by a Ca 2+ spark in the absence of EGTA (Cheng et al, 1993;Nelson et al, 1995), and B of 80 (Guerrero et al, 1994). With these conditions, a Ca 2+ flux of 1.3 × 10 17 mol/s (equivalent to a current of 2.5 pA) would produce a [Ca 2+ ] i of 50 nM.…”

Axonal Neuropathy-associated TRPV4 Regulates Neurotrophic Factor-derived Axonal Growth

“…Gain-of-function mutants such as R269H and R316C elicit increased current responses to TRPV4 agonists as well as Ca 2ϩ loading to cells. These Ca 2ϩ overloads caused by constitutive activities of TRPV4 mutants are believed to be a leading cause of neurodegeneration of peripheral nerves (6,9). Although the association between TRPV4 mutation and motor/sensory neuropathies has been clearly established, the physiological function of TRPV4 in sensory and motor neurons in the normal condition remains unknown.…”

“…Recent genetic linkage studies in patients indicate that mutations in the ankyrin repeat domains of TRPV4 are implicated in the neuropathies (5)(6)(7)(8). The gain-of-function mutations in the ankyrin repeat domain of TRPV4 are known to cause degenerative diseases involving peripheral nerves (6,7,9). Gain-of-function mutants such as R269H and R316C elicit increased current responses to TRPV4 agonists as well as Ca 2ϩ loading to cells.…”