Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations

Abstract

TRPV4 is a mechanically activated Ca(2+)-passing channel implicated in the sensing of forces, including those acting on bones. To date, 33 mutations are known to affect human bone development to different extents. The spectrum of these skeletal dysplasias (SD) ranges from dominantly inherited mild brachylomia (BO) to neonatal lethal forms of metatropic dysplasia (MD). Complexities of the results from fluorescence and electrophysiological studies have led to questions on whether channel activity is a good predictor of disease severity. Here we report on a systematic examination of 14 TRPV4 mutant alleles covering the entire SD spectrum. Expressed in Xenopus oocyte and without any stimulation, the wild-type channel had a ~1% open probability (Po) while those of most of the lethal MD channels approached 100%. All mutant channels had higher basal open probabilities, which limited their further increase by agonist or hypotonicity. The magnitude of this limitation revealed a clear correlation between the degree of over-activity (the molecular phenotype) and the severity of the disease over the entire spectrum (the biological phenotype). Thus, while other factors are at play, our results are consistent with the increased TRPV4 basal activity being a critical determinant of the severity of skeletal dysplasia. We discuss how the channel over-activity may lead to the "gain-of-function" phenotype and speculate that the function of wild-type TRPV4 may be secondary in normal bone development but crucial in an acute process such as fracture repair in the adult.

Figure 1. All fourteen mutant TRPV4 cRNAs tested generated robust currents in Xenopus oocytes.

Two to four days after the injection of 22 ng of wild-type or lesser amounts of mutant cRNAs, oocytes were examined with a two-electrode voltage clamp, held at −60 mV and tested every 5 sec between −100 and +60 mV for 1 sec. All mutants retained strong rectification against steady-state inward current. See text. All calibration bars are 4 µA×0.2 sec.

Figure 2. Basal current densities of oocytes expressing TRPV4 mutant channels are greater than those of wild type but highly variable.

Peak currents at +60 mV from oocytes expressing wild type or mutant TRPV4 assessed between 72 and 96 hours after cRNA injection were measured and standardized to the amount of cRNA injected (triangles plotted on a log scale because of the large range, mean ± s.e.m.). Alleles are grouped by the severity of disease they cause. “WT” denotes wild-type and disease abbreviations are described in the text. MD mutants are further subdivided into mild, moderate (“mod”), severe (“sev”) and infantile/neonatal lethal (“lethal”) as described in Camacho et al. (2010) . Whereas the expression of the more severe mutants often reached peak within 24 hours, those of weaker alleles generally took longer and unstimulated wild-type currents were usually not apparent until 72 hours after injection.

Figure 3. Mutant channels have limited hypotonic responses that inversely correlate with clinical severity.

a. Currents were monitored from an oocyte expressing wild-type TRPV4 with 250 ms depolarizations to +40 mV from a hold of 0 mV every 10 s after the removal of 100 mM sorbitol from the media (left, with serial depolarizations vertically stacked). Peak currents from serial depolarizations are plotted against hypotonic exposure time on right. Vertical current scale for both raw traces and plot is shown on right. b. Same as in a, except that oocyte is expressing the neonatal-lethal allele L618P and the test potential was +30 mV. c. Relative increases in peak currents caused by hypotonicity from oocytes expressing wild type and mutant TRPV4 channels assessed at +60 mV. Alleles are grouped by the severity of the dysplasia they cause as described in Fig. 2 legend. Triangles represent individual data points from unique oocytes, vertical bars are the allele means, and error bars are the standard error within each allele. d. Average of the individual allele average hypotonic responses grouped by clinical severity, with error bars representing the s.e.m. of the allele averages. “m/m MD” represents mild and moderate MD, whereas “s/l” represents severe and lethal. Whereas the clinically mildest BO mutants only have a moderate reduction in their response to hypotonicity, most of the severest MD mutants have little or no response.

Figure 4. The limitation of the very large GSK responses shows an even more pronounced negative correlation with disease severity.

Responses to 1 µM GSK are shown. All figures are arranged as in Fig. 3. Note the near 100-fold increase of the wild-type current here (a, c, d) as compared to the <10-fold increase by hypotonicity. As with hypotonicity, the majority of the most severe mutants have little or no response to GSK (b, c, d).

Figure 5. Single channel analysis confirms that lack of response to stimuli is due to saturated basal open probability in I604M.

Excised patches with single active channels were analyzed from oocytes expressing either wild-type cRNA (a, n>100) or the neonatal-lethal allele I604M (b, n = 3). Shown are outward unitary currents at +50 mV before (left) and after (right) brief exposure to 1 µM GSK. Open (o) and closed (c) current levels are marked. 50-fold time expansions are shown in B to confirm that the downward spikes indeed reflect the rare full channel closure as evidenced by occasional brief plateaus at the fully closed level (*).