P-3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na+,K+-ATPase

P-3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investiga ted for its application as a phototrigger for the rapid activation of elect rogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer l aser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the ph otolytic yields of P-3-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P-3 -[1,2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. U sing time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-cage d ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na+,K+-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na+,K+-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the e lectrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. it is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compoun d are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6.0) conditions, pHP-cag ed ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.