Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Acceptor-donor relationships in the transglutaminase-mediated cross-linking of lens beta-crystallin subunits

Following the isolation of the N epsilon-(gamma-glutamyl)lysine-containing polymers from human cataracts, our efforts were directed to induce such cross-links experimentally in rabbit lens, and evidence was obtained for the selective reactivities of certain beta-crystallin subunits in this transglutaminase-catalyzed event. In the present work, we examined the enzymatic cross-linking of purified crystallins individually (alpha, beta H, beta L, and gamma) and in combinations, with particular emphasis on forming the approximately 55K dimer. This species was the primary product in the cross-linking of beta H-crystallins; beta L also reacted with transglutaminase. Neither alpha- nor gamma-crystallins formed appreciable amounts of cross-linked structures with transglutaminase. Dansylcadaverine, known to compete against the reactive lysines of proteins in forming N epsilon-(gamma-glutamyl)lysine cross-bridges, was shown to inhibit the generation of dimeric and higher ordered oligomers from beta H and beta L. The fluorescent amine specifically labeled only two subunits in beta H (approximately 29-30K and approximately 26K) and one in beta L (approximately 26K), identifying these substrates as possessing transglutaminase-reactive endo-gamma-glutaminyl residues. An antiserum to bovine beta Bp recognized the approximately 23K subunit of rabbit beta-crystallins and also the approximately 55K dimer, suggesting that the approximately 23K protein participates as a lysine donor in generating the cross-linked dimer with transglutaminase. Inasmuch as the same antiserum reacts with an approximately 50K material reported to appear in increasing amounts with age in human lens, the results lend added support to the physiological significance of transglutaminase in the aging of lens.     

Light and electron microscopical localization of concanavalin A lectin binding sites in rat epiphyseal chondrocytes

Summary Concanavalin A lectin binding sites have been detected within the cytoplasm of epiphyseal chondrocytes. Correlative light and electron microscopic results were obtained, indicating the presence of-d-mannose and/or -D-glucose residues detected by the lectin in the rough endoplasmic reticulum region. Quantitation of the electron microscopic cytochemical reaction also showed that the specific labelling was almost exclusively localized in the lumen of endoplasmic reticulum cisternae. No significant staining was found in other membrane compartments or extracellular matrix. This labelling pattern could be considered as the cytochemical evidence ofN-glycosylation processes occurring during the biosynthesis of cartilage extracellular matrix components by chondrocytes.     

Interspecific variation in arrangement and morphology of micrasters of Tethya species (Porifera,Demospongiae)

Summary A new distinctive feature between the two Mediterranean species of Tethya, T. aurantium and T. citrina has been found in the body arrangement of different types of micrasters. Contrary to the previous assumptions, T. aurantium has two clearly distinct categories of micrasters: the chiaster-tylaster in the cortex and the larger, slender oxyaster in the choanosome. T. citrina has only slightly differentiated micraster sets in the cortex and choanosome; in the latter the shape of micrasters is close to that of oxyasters. SEM analysis shows that differences in micraster shape depend on the cylindrical or conical form of rays and on the distribution, density and strength of the microspines along their axis. The relationship between the degree of micraster differentiation and the development of the cortex in the two species is discussed.     

Experimental study of the influence of errors of genetic correlation estimates on unrestricted,optimum, and desired gains selection indices

Summary Effects of errors in estimates of the genetic correlation on the accuracy of unrestricted, optimum, and desired gains selection indices were examined experimentally in Tribolium castaneum. Three lines were selected for three generations for pupal weight at 21 days and adult weight at 31 days, using unrestricted (I9), optimum (O9), and desired gains (G9) index selection methods. The genetic correlation between pupal and adult weights in the base population was 0.95. The optimum index was designed to set the response of pupal weight by a fixed amount, while in the desired gains index the responses of pupal and adult weights were specified as being equal to 31. Three other indices were constructed using a deliberately incorrect genetic correlation (0.25), i.e., unrestricted (I2), optimum (O2), and desired gains (G2). Responses observed in unrestricted index lines (I9 versus I2) and optimum index lines (O9 versus O2) did not differ significantly, even though lines I9 and I2 differed in a practical sense. Responses in desired gains index lines (G9 versus G2) differed significantly. Responses obtained for aggregate genotype (pupal weight + adult weight) and for the component traits were greater in line I9 than those obtained in line I2. Responses obtained in the O9 and O2 lines for pupal and adult weights were similar, while those obtained in the G9 and G2 lines were similar for pupal weight but not (P<0.05) for adult weight. Therefore, underestimation of the genetic correlation seems to affect the efficiency of a desired gains index more than that of unrestricted or optimum indices.