G protein activated inward-rectifying K⁺ (GIRK) channels are expressed in the heart, in various neurones and endocrine cells. They are activated by direct interaction of their subunits with ßg subunits released from heterotrimeric G proteins upon agonist stimulation of appropriate G protein coupled receptors (GPCRs). Their opening causes slow IPSPs, heart rate reduction and important roles of GPCR-GIRK signalling in pain sensation and drug addiction are discussed. Upon exposure to an agonist, activation of GIRK current is followed by a rapid decay within seconds, termed fast or acute desensitization, which is unique to the receptor–GIRK channel pathway. Although it is commonly accepted that this type of desensitization is localized downstream of the receptor, the underlying mechanism is still a matter of debate. Whereas some authors suggested fast desensitization to reflect a property of the G protein cycle (e.g. Chuang et al., 1998), data from our lab provided evidence that it is caused by a current-related reduction in the electrochemical driving force for K⁺ ions (Bender et al., 2004). In order to distinguish between these two mechanisms, kinetics of interactions of signalling proteins expressed in HEK293 cells relevant to this pathway were measured using combined FRET-microscopy and patch clamp-recordings. Different interacting partners were expressed as CFP/YFP fusion constructs to measure agonist-evoked dynamic interactions using FRET. FRET signals were compared to simultaneously recorded GIRK currents activated via co-expressed A₁ adenosine receptors. Rapid G protein activation could be recorded using G_ai-YFP and G_ß2-CFP. However, no time-dependent change in FRET corresponding to fast desensitization of the GIRK current could be detected, supporting the notion that fast desensitization is unrelated to kinetics of the G protein cycle. The activation of GIRK1/4 channels by ßg subunits released from heterotrimeric G proteins was investigated in terms of fast desensitization using G_ß1-CFP and GIRK4-YFP/GIRK1 or GIRK1-YFP/GIRK4. No fast desensitization could be detected both in the GIRK current and in the corresponding FRET ratio. This is due to the formation of GIRK4 homotetramers, which are much slower than GIRK1/4 channels and therefore lack a fast desensitization component.

A tandem construct (GIRK1-CFP-GIRK4) was generated to avoid homotetrameric GIRK4 channels and to assure a 1:1 stoichiometry of channel subunits. Expression of this construct results in agonist-induced currents with rapid activation. Experiments to study its kinetic properties using simultaneous FRET- and patch clamp-recordings are under way.