PARTIAL AGONISTS AND ANTAGONISTS REVEAL A 2ND PERMEABILITY STATE OF HUMAN LYMPHOCYTE P2Z P2X(7) CHANNEL/

Extracellular ATP is known to trigger apoptosis of thymocytes and lymp hocytes through a P2Z receptor at which ATP is a partial agonist, givi ng only 70% of the maximum response of 3'-O-(4-benzoyl)benzoyl-adenosi ne 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor an d its associated ion channel are Ca2+ (and Ba2+) selective but also pa ss molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X(7) gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and Ba-133(2+), immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resol ved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioad enosine 5'-triphosphate, or adenosine 5'-O-(3-thiotriphosphate). Maxim ally effective concentrations of BzATP (50 mu M) induced immediate upt ake of ethidium at a rate linear with time. In contrast, a delay was o bserved (30 s) before ethidium uptake commenced after addition of maxi mally effective ATP concentrations (500 mu M) at 37 degrees C, and the delay was longer at 24 degrees C. ATP addition 2-10 min before ethidi um abolished the delay. The delay was longer with other partial agonis ts and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10-20 mu M). P2 Z/P2X(7) inhibitors, KN-62 and 5-(N,N-hexamethylene)-amiloride, reduce d the rate of agonist-induced ethidium uptake and lengthened the delay . The results support a model in which agonists for P2Z/P2X(7) recepto r mediate an immediate channel opening allowing passage of small inorg anic cations, followed by a slow further permeability increase allowin g passage of larger permeant cations like ethidium. The rate of the se cond step depends on time and temperature and the efficacy and concent ration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X(7) channels held in the initial open state.