Long mutant dystrophins and variable phenotypes: evasion of nonsense-mediated decay?

More than 98% of Duchenne muscular dystrophy (DMD) mutations result in the premature termination of the dystrophin open reading frame at various points over its 11-kb length. Despite this wide variation in coding potential (0%-98.6% of the full-length protein), the truncating mutations are associated with a surprisingly uniform severity of phenotype. This uniformity is probably attributable to ablation of the message by nonsense-mediated decay (NMD). The rare truncating mutations that occur near the 3' end of the dystrophin gene (beyond exon 70) can however result in extremely variable phenotypes (both intra- and inter-familially). We suggest that all proteins encoded by such mutant genes are capable in principle of rescuing the DMD phenotype but that NMD abrogates the opportunity to effect this rescue. The observed variability may therefore reflect an underlying variation in the efficiency of NMD between individuals. We discuss this hypothesis with particular reference to a well-characterised Becker muscular dystrophy patient with a frameshift mutation, where expression of a truncated dystrophin rescues the muscular but not mental phenotype.

Autoradiographic demonstration of quinuclidinyl benzilate binding sites in the vestibular organs of the gerbil.

Gerbil vestibular tissues were isolated by microdissection and incubated in vitro with 3H-quinuclidinyl benzilate (3H-QNB). Control tissues were incubated in medium containing unlabeled atropine to differentiate non-specific from specific binding. Autoradiographic grain densities were determined by morphometric techniques and evaluated by two-tailed t-test. The label densities of sensory epithelia from experimental preparations of ampulla, utricle and saccule were found to be significantly higher than those in the adjacent endolymphatic compartment and also higher than those of adjacent stromal tissue comprising connective tissue, nerve fibers and capillaries. In contrast, no tissue region in atropine controls showed label density significantly above that of the endolymphatic compartment. Label density of ampullar sensory epithelium incubated with 3H-QNB alone was significantly higher than that of sensory epithelium from utricle or saccule. Grain density was greater in the peripheral regions of the ampullar crista compared to the vertex. Appreciable label was also present in nerve bundles beneath the sensory epithelium of the ampulla. The current study demonstrates the existence of putative muscarinic neurotransmitter/neuromodulator receptor sites in mammalian vestibular sense organs at locations corresponding to efferent innervation, with particularly significant concentrations in the ampulla.

Aldosterone mediates an increase in [3H]ouabain binding at Na+, K(+)-ATPase sites in the mammalian inner ear.

Na+,K(+)-ATPase has been implicated in the maintenance of high [K+], low [Na+] in endolymph of the inner ear, ionic properties considered to support transduction by the receptor cells. In exocrine ion-transporting epithelia, Na+,K(+)-ATPase activity is modulated by aldosterone, a mineralocorticoid hormone. In the present study, the effect of alteration of serum aldosterone levels on Na+,K(+)-ATPase in ion-transporting regions of the mammalian inner ear was investigated. A high Na+/low K+ diet offered ad libitum for 5 days was utilized to significantly decrease serum aldosterone in male Hartley guinea pigs compared to controls. An injection of aldosterone (10 micrograms/100 g b.wt.) 21 h prior to sacrifice resulted in significant elevation of serum aldosterone over that obtained with the high Na+/low K+ diet. Binding of [3H]ouabain, a specific inhibitor of Na+,K(+)-ATPase, was significantly elevated in microdissected lateral wall of the basal turn of the cochlea and in the ampulla of the semicircular canal, for aldosterone-injected vs. vehicle-injected animals. Serum [Na+] and [Cl-] were elevated in animals on the high Na+/low K+ diet and unaltered by administration of exogenous aldosterone. The enhancement of ouabain binding in inner ear tissues observed in aldosterone-injected animals, therefore, did not appear to reflect an alteration of serum electrolytes per se. The results of these experiments are consistent with the hypothesis that aldosterone increases the number of Na+,K(+)-ATPase sites in ion-transporting epithelia of the mammalian cochlea and semicircular canal.

Ultrastructural localization of Na+/K(+)-ATPase in rodent olfactory epithelium.

The olfactory epithelium is comprised of bipolar sensory neurons, sustentacular cells, and basal cells. The sensory neurons have apical knobs and cilia, which project into the olfactory mucus toward the nasal lumen, and represent presumptive sites of odorant binding. Ionic currents, measured across this epithelium in both the resting and odorant-stimulated states, are known to be sustained, at least in part, by active transport of sodium. Information identifying the cellular sites of ion transport in olfactory sensory epithelium will therefore aid in elucidating the ionic mechanisms associated with olfactory transduction. The membrane-bound enzyme Na+/K(+)-ATPase mediates active ion transport in many other cells and tissues. We have consequently employed the cytochemical technique reported by Ernst (J. Histochem. Cytochem., 20 (1972) 23-38, 1322) to identify possible sites of elevated Na+/K(+)-ATPase activity in olfactory epithelium. This procedure detects inorganic phosphate (Pi) released from an artificial substrate (nitrophenyl phosphate) by enzyme catalytic activity. In the presence of strontium ion. Pi is precipitated near regions of high enzymatic activity, then converted to a product visible in the electron microscope. Parallel control preparations were incubated in media (1) supplemented with the specific Na+/K(+)-ATPase inhibitor ouabain (to abolish formation of specific reaction product); (2) with substrate deleted (to demonstrate possible non-specific binding of Sr2+ and/or Pb2+); or (3) with the necessary cofactor K+ deleted. In tissues incubated for demonstration of Na+/K(+)-ATPase activity, reaction product was associated with apical knobs, cilia and dendrites of olfactory receptor neurons at the apical surface. In the more proximal region of the epithelium, reaction product was associated with cell bodies and axons of the sensory neurons, and with the lateral membranes of sustentacular cells. Reaction product was deposited intracellularly, compatible with the known mechanism of the Na+/K(+)-ATPase enzymatic reaction. In control specimens incubated with ouabain, with substrate deleted, or with K+ deleted, only a small quantity of non-specific precipitate was observed. These results are discussed with reference to the various sodium currents implicated in olfactory transduction and transepithelial transport.

Protein associated with the sensory cell layer of the rainbow trout saccular macula.

A protein has been detected that is associated with the saccular hair cell layer of the rainbow trout, Salmo gairdnerii R. By one- and two-dimensional SDS polyacrylamide gel electrophoresis, the molecular weight and isoelectric point of this protein are estimated to be 13.6 and 8.8 kDa, respectively. The 13.6 kDa protein cannot be detected electrophoretically in brain, gill, liver, and fractions containing the basal lamina, non-sensory epithelium, and saccular nerve. This protein does not bind antibodies to bovine myelin basic protein, while trout myelin basic proteins in the same molecular weight range do. In addition, the protein does not bind concanavalin A or react with the periodic acid-Schiff reagent. The 13.6 kDa band represents about 1% of the total protein in saccular sensory epithelium, and may be a marker protein for the hair cell layer.

Possible functional roles of Na+,K+-ATPase in the inner ear and their relevance to Ménière's disease.

This article reviews the functions of the enzyme Na+,K+-ATPase in epithelial tissues and discusses early and recent biochemical, physiologic and morphologic studies of the enzyme in the inner ear. The purpose of the investigation was to learn whether a relationship between perturbations in activity of the enzyme and Ménière's disease is possible. It is concluded that the preponderance of the evidence indicates that Na+,K+-ATPase plays a role in regulating ion transport into the scala media, but that the significance of the distribution of the enzyme along only one cell type (the marginal) in the functional chains of cells of the outer cochlear wall needs further study. The possible vasoconstrictive effects of ouabain perfusions employed by some investigators must also be taken into account. Recent cytochemical and autoradiographic studies have demonstrated high levels of Na+, K+-ATPase on cochlear nerve fibers, especially near the foramina nervosa and within the organ of Corti. Thus, perturbations in Na+,K+-ATPase activity in the inner ear not only could affect certain aspects of fluid balance, but also could account for the sensory disturbances experienced by patients who have Ménière's disease.

Cellular localization of Na+,K+-ATPase in the mammalian cochlear duct: significance for cochlear fluid balance.

Cytochemical and autoradiographic procedures were employed to determine the cellular distribution of Na+,K+-ATPase in the guinea pig cochlea. The highest activity was associated with the stria vascularis and was restricted almost entirely to the contraluminal extensions of the marginal cells. Elevated levels of activity were also observed in stromal cells of the spiral prominence, external sulcus, and spiral limbus. The pattern of activity in the latter tissues was unusual in being symmetrically distributed along the plasma membranes of the reactive cells. In the organ of Corti, only neural elements showed appreciable activity. Possible functions of the enzyme are discussed in relation to cochlear fluid balance.