Distinguishing surface effects of calcium ion from pore-occupancy effects in Na+ channels

The effects of calcium ion on the Na+ activation gate were studied in squid giant axons. Saxitoxin (STX) was used to block ion entry into Na+ channels without hindering access to the membrane surface, making it possible to distinguish surface effects of calcium from pore-occupancy effects. In the presence of STX, gating kinetics were measured from gating current (Ig). The kinetic effects of external calcium concentration changes were small when STX was present. In the absence of STX, lowering the calcium concentration (from 100 to 10 mM) slowed the closing of Na+ channels (measured from INa tails) by more than a factor of 2. Surprisingly, the voltage sensitivity of closing kinetics changed with calcium concentration, and it was modified by STX. Voltage sensitivity apparently depends in part on the ability of calcium to enter and block the channels as voltage is driven negative. In external medium with no added calcium, INa tail current initially increases in amplitude severalfold with the relief of calcium block, then progressively slows and gets smaller, as calcium diffuses out of the layers investing the axon. INa tails seen just before the current disappears suggest that closing in the absence of channel block is very slow or does not occur. INa amplitude and kinetics are completely restored when calcium is returned. The results strongly suggest that calcium occupancy is a requirement for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation.

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.

Light-Induced Changes in Hydrogen, Calcium, Potassium, and Chloride Ion Fluxes and Concentrations from the Mesophyll and Epidermal Tissues of Bean Leaves. Understanding the Ionic Basis of Light-Induced Bioelectrogenesis1

Noninvasive, ion-selective vibrating microelectrodes were used to measure the kinetics of H+, Ca2+, K+, and Cl− fluxes and the changes in their concentrations caused by illumination near the mesophyll and attached epidermis of bean (Vicia faba L.). These flux measurements were related to light-induced changes in the plasma membrane potential. The influx of Ca2+ was the main depolarizing agent in electrical responses to light in the mesophyll. Changes in the net fluxes of H+, K+, and Cl− occurred only after a significant delay of about 2 min, whereas light-stimulated influx of Ca2+ began within the time resolution of our measurements (5 s). In the absence of H+ flux, light caused an initial quick rise of external pH near the mesophyll and epidermal tissues. In the mesophyll this fast alkalinization was followed by slower, oscillatory pH changes (5–15 min); in the epidermis the external pH increased steadily and reached a plateau 3 min later. We explain the initial alkalinization of the medium as a result of CO2 uptake by photosynthesizing tissue, whereas activation of the plasma membrane H+ pump occurred 1.5 to 2 min later. The epidermal layer seems to be a substantial barrier for ion fluxes but not for CO2 diffusion into the leaf.

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca2+-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca2+]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca2+]cyt and inhibition of growth that was similar to the effect of the Ca2+ channel blocker La3+ and the Ca2+ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca2+ channel blockers verapamil and nifedipine did not induce a decrease in [Ca2+]cyt in these cells and also failed to inhibit growth. Al and La3+, but not verapamil or nifedipine, reduced the rate of Mn2+ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca2+- and Mn2+-permeable channels. These results suggest that Al may act to block Ca2+ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

Ion balance in waters through inductively coupled plasma optical emission spectrometry

Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) has been employed to carry out the determination of both major anions and cations in water samples. The anion quantification has been performed by means of a new automatic accessory. In this device chloride has been determined by continuously adding a silver nitrate solution. As a result solid silver chloride particles are formed and retained on a nylon filter inserted in the line. The emission intensity is read at a silver characteristic wavelength. By plotting the drop in silver signal versus the chloride concentration, a straight line is obtained. As regards bicarbonate, this anion has been on-line transformed into carbon dioxide with the help of a 2.0 mol L−1 nitric acid stream. Carbon signal is linearly related with bicarbonate concentration. Finally, information about sulfate concentration has been achieved by means of the measurement of sulfur emission intensity. All the steps have been simultaneously and automatically performed. With this setup detection limits have been 1.0, 0.4 and 0.09 mg L−1 for chloride, bicarbonate and sulfate, respectively. Furthermore, it affords good precision with RSD below 6 %. Cation (Ca, Mg, Na and K) concentration, in turn, has been obtained by simultaneously reading the emission intensity at characteristic wavelengths. The obtained limits of detection have been 8 × 10−3, 2 × 10−3, 8 × 10−4 and 10−2 mg L−1 for sodium, potassium, magnesium and calcium, respectively. As regards sample throughput, about 30 samples h−1 can be analysed. Validation results have revealed that the obtained concentrations for these anions are not significantly different as compared to the data provided by conventional methods. Finally, by considering the data for anions and cations, precise ion balances have been obtained for well and mineral water samples.