title: Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D018942:Macrolides; C057620:bafilomycin A; D009249:NADP; C024376:NAADP; D006180:Proton-Translocating ATPases; D002118:Calcium