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.     

Molecular Characterization of Larval Peripheral Thermosensory Responses of the Malaria Vector Mosquito Anopheles gambiae

Thermosensation provides vital inputs for the malaria vector mosquito, Anopheles gambiae which utilizes heat-sensitivity within a broad spectrum of behaviors, most notably, the localization of human hosts for blood feeding. In this study, we examine thermosensory behaviors in larval-stage An. gambiae, which as a result of their obligate aquatic habitats and importance for vectorial capacity, represents an opportunistic target for vector control as part of the global campaign to eliminate malaria. As is the case for adults, immature mosquitoes respond differentially to a diverse array of external heat stimuli. In addition, larvae exhibit a striking phenotypic plasticity in thermal-driven behaviors that are established by temperature at which embryonic development occurs. Within this spectrum, RNAi-directed gene-silencing studies provide evidence for the essential role of the Transient Receptor Potential sub-family A1 (TRPA1) channel in mediating larval thermal-induced locomotion and thermal preference within a discrete upper range of ambient temperatures.     

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.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.