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.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.     

Role of Activity of Gastrointestinal Microflora in Absorption of Calcium and Magnesium in Rats Fed β1-4 Linked Galactooligosaccharides

Rats fed a diet containing β1-4 linked galactooligosaccharides (GOS) (5 g/100 g of diet) absorbed calcium and magnesium more efficiently than those fed the control diet. However, the increment obtained through GOS-feeding was reduced by neomycin sulfate 0.67 g/100 g of diet). Since the decrease in cecal pH in rats fed GOS was suppressed by neomycin-feeding, bacterial action in the digestive tract was considered to be reduced by neomycin-feeding. Our findings suggest that the action of intestinal bacteria is necessary for the effects of GOS.     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Mercury in blood cells—Altered elemental profiles

The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure.     

Application of PIXE analysis to the study of the regional distribution of trace elements in normal human brain

A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se, and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed matter brain areas.     

Effects of essential divalent metals on carcinogenicity and metabolism of nickel and cadmium

Interactions between the physiologically essential metals calcium, magnesium, and zinc and the carcinogenic metals nickel and cadmium were investigated to help elucidate the mechanisms of action of the carcinogenic metals. Bioassay studies revealed several significant findings, including: (1) the ability of magnesium and calcium to inhibit nickel-induced elevation of pulmonary adenoma incidence in strain A mice; (2) the ability of magnesium, but not of calcium, to prevent cadmium-induced subcutaneous sarcoma formation; and (3) the ability of magnesium, but not of calcium, to inhibit nickel-induced muscle tumor formation. Biochemical studies indicated a direct relationship between the antitumorigenic potential of magnesium and the capacity of this metal to: (1) inhibit nickel and cadmium uptake by the target tissues in vivo; (2) inhibit nickel-induced disturbances in DNA synthesis in vivo; (3) inhibit nuclear and cytosolic uptake of nickel by the target tissue cells in vivo; and (4) inhibit nickel and cadmium binding to DNA in vitro. Calcium, which in most cases did not prevent carcinogenesis, had no consistent influence on the uptake of carcinogenic metals or their biochemical effects in the target tissues. Magnesium and zinc, but not calcium, were also found to attenuate the acute toxic effects of nickel, indicating a possible correlation between prevention of acute effects and reduction in tumorigenicity. Zinc, which antagonizes cadmium tumorigenicity in the rat testis, was found to reduce markedly cadmium uptake into isolated testicular interstitial cells. Also, zinc was found to inhibit strongly cadmium binding to DNA in vitro.     

ARAGONITE CRYSTALS IN SPIROGYRA SP. (CHLOROPHYTA)

Intracellular crystals of aragonite have been identified by selected area electron diffraction (SAED) in a species of the freshwater filamentous alga Spirogyra from the Thames River, Ontario, Canada. The crystals are 2 to 24 μm in diameter, and characterized by a unique cross-shaped morphology, in which needle-like, or prismatic outgrowths develop from a common axis. Crystals may be dispersed throughout filaments, but tend to cluster as aggregates towards the centre .