Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-32P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca2+ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al3+, H+, or Na+ (or other cationic toxicants), supplementation of the medium with higher levels of Ca2+ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca2+ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca2+-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca2+ at the cell surface if the surface Ca2+ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca2+ beyond mechanisms I and II, and may involve Ca2+-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na+ toxicity, moderately in the alleviation of H+ toxicity, and not at all in the alleviation of Al3+ toxicity.

Investigating responses of calcium carbonate nucleation rates in artificial seawater to changes in saturation states due to ocean acidification

Senior thesis written for Oceanography 445

Studies of the interaction of Potassium(I), Calcium(II), Magnesium(II), and Copper(II) with Cyclosporin A

Metal ion binding properties of the immunosuppressant drug cyclosporin A have been investigated. Complexation studies in acetonitrile solution using H-1 NMR and CD spectroscopy yielded 1:1 metal-peptide binding constants (log(10)K) for potassium(l), < 1, magnesium(II), 4.8 +/- 0.2. and calcium(II), 5.0 +/- 1.0. The interaction of copper(II) with cyclosporin A in methanol was investigated with UV/visible and electron paramagnetic resonance (EPR) spectroscopy. No complexation of copper(II) was observed in neutral solution. In the presence of base, monomeric copper(II) complexes were detected. These results support the possibility that cyclosporin A has ionophoric properties for biologically important essential metal ions. (C) 2003 Elsevier Inc. All rights reserved.

Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release. © 1995 Academic Press. All rights reserved.

Controlling particle size in the Stöber process and incorporation of calcium

The Stӧber process is commonly used for synthesising spherical silica particles. This article reports the first comprehensive study of how the process variables can be used to obtain monodispersed particles of specific size. The modal particle size could be selected within in the range 20 – 500 nm. There is great therapeutic potential for bioactive glass nanoparticles, as they can be internalised within cells and perform sustained delivery of active ions. Biodegradable bioactive glass nanoparticles are also used in nanocomposites. Modification of the Stӧber process so that the particles can contain cations such as calcium, while maintaining monodispersity, is desirable. Here, while calcium incorporation is achieved, with a homogenous distribution, careful characterisation shows that much of the calcium is not incorporated. A maximum of 10 mol% CaO can be achieved and previous reports are likely to have overestimated the amount of calcium incorporated.

The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.