Regulation of the cardiac Na (�) -Ca 2� exchanger by Ca 2� . Mutational analysis of the Ca (2�) -binding domain

The sarcolemmal Na+-Ca 2+ exchanger is regulated by intracellular Ca ~+ at a high affinity Ca 2+ binding site separate from the Ca ~+ transport site. Previous data have suggested that the Ca 2+ regulatory site is located on the large intracellular loop of the Na +-Ca 2+ exchange protein, and we have identified a highaffinity 4SCa2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca ~+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca ~+ regulation was measured as the stimulation of reverse Na+-Ca ~+ exchange (intracellular Na + exchanging for extracellular Ca ~+) by intracellular Ca ~+. Single-site mutations within two acidic clusters of the Ca 2+ binding domain lowered the apparent Ca ~+ affinity at the regulatory site from 0.4 to 1.1-1.8 ~.M. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca~

Well-Known Mediators of Selective Oxidation with Unknown Electronic Structure: Metal-Free Generation and EPR Study of Imide‑<i>N</i>‑oxyl Radicals

Nitroxyl radicals are widely used in chemistry, materials sciences, and biology. Imide-<i>N</i>-oxyl radicals are subclass of unique nitroxyl radicals that proved to be useful catalysts and mediators of selective oxidation and CH-functionalization. An efficient metal-free method was developed for the generation of imide-<i>N</i>-oxyl radicals from <i>N</i>-hydroxyimides at room temperature by the reaction with (diacetoxyiodo)­benzene. The method allows for the production of high concentrations of free radicals and provides high resolution of their EPR spectra exhibiting the superhyperfine structure from benzene ring protons distant from the radical center. An analysis of the spectra shows that, regardless of the electronic effects of the substituents in the benzene ring, the superhyperfine coupling constant of an unpaired electron with the distant protons at positions 4 and 5 of the aromatic system is substantially greater than that with the protons at positions 3 and 6 that are closer to the <i>N</i>-oxyl radical center. This is indicative of an unusual character of the spin density distribution of the unpaired electron in substituted phthalimide-<i>N</i>-oxyl radicals. Understanding of the nature of the electron density distribution in imide-<i>N</i>-oxyl radicals may be useful for the development of commercial mediators of oxidation based on <i>N</i>-hydroxyimides