Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones

Abstract

The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P³-ester derivatives of guanosine 5′-0(3-thio)triphosphate (caged GTP-γ-S) and 5′-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-γ-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-γ-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5–10 min, and a single pulse of 300–350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30–120 s following photochemical release of approximately 20μM GTP-gg-S. The peak calcium channel current was reduced by about 70% with a slow time course [t _1/2 1.5±0.2 min (mean±SEM);n=5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP. These results suggest that the channels underlying the transient and the sustained components of the whole cell current may be differentially molulated by GTP analogues.