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.     

Plant phosphoglucose isomerase genes lack introns and are expressed in Escherichia coli

Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.     

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.     

Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.