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

Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state

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