Increased Basal Activity Is a Key Determinant in the Severity of Human Skeletal Dysplasia Caused by TRPV4 Mutations

TRPV4 is a mechanically activated Ca2+-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

Carboxyl tail prevents yeast K(+) channel closure: proposal of an integrated model of TOK1 gating.

TOK1 encodes the channel responsible for the prominent outward K(+) current of the yeast plasma membrane. It can dwell in several impermeable states, including a rapidly transiting, K(+)-electromotive-force-dependent "R" (rectifying) state, a voltage-independent "IB" (interburst) state, and a set of [K(+)](ext) and voltage-dependent "C" (closed) states. Whereas evidence suggests that the C states result from the constriction of an inner gate at the cytosolic end of the pore, R is most likely an intrinsic gating property of the K(+) filter. Here, we present evidence that Tok1's carboxyl-tail domain also plays an intimate role in channel gating by dynamically preventing inner-gate closures. We present an integrated model of TOK1 gating in which the filter gate, inner gate, and carboxyl tail interact to produce the various phenomenological states. Both wild-type and tailless behaviors can be replicated using Monte Carlo computer simulations based on this model

Basal current densities of oocytes expressing <i>TRPV4</i> mutant channels are greater than those of wild type but highly variable.

<p>Peak currents at +60 mV from oocytes expressing wild type or mutant <i>TRPV4</i> 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 <i>et al.</i> (2010) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019533#pone.0019533-Camacho1" target="_blank">[3]</a>. 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.</p

All fourteen mutant <i>TRPV4</i> cRNAs tested generated robust currents in Xenopus oocytes.

<p>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.</p

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

<p>Responses to 1 µM GSK are shown. All figures are arranged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019533#pone-0019533-g003" target="_blank">Fig. 3</a>. 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).</p