Physiological and molecular characterization of an IRK-type inward rectifier K⁺ channel in a tumour mast cell line

Abstract

The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, I _KIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this K_IR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K⁺ inward rectifiers of the IRK type. Channel gating was rapid, first order (τ ≈ 1 ms at −100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K⁺ concentration ([K⁺]_o) and inward rectification persisted in the absence of internal Mg²⁺. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na⁺, Cs⁺ and Ba²⁺. Initial I _KIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5′-Q-(3-thiotriphosphate) (GTP[γS]) suppressed I _KIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 μM preloaded GTP[γS]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)⁺ RNA identified a full length K⁺ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K⁺ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.