Dynamic interactions of signalling proteins can be measured using FRET-microscopy. The vast majority of studies using this technique have been performed using cell lines as heterologous expression system. In the present study we measured interactions between various components of the receptor - Gi/o - GIRK channel pathway in cultured (4-5 d) adult cardiac myocytes, i.e. on a cell-type specific background. CFP / YFP tagged pairs of signalling proteins were expressed using adenoviral gene transfer. Functional efficiency of the fusion constructs was verified using GIRK channels as readout. Upon activation of the A1-receptor positive changes in FRET were observed between A1-R and Gb. Confirmatory to recent publications, a positive dynamic FRET signal was obtained from Gb-CFP and Gai-YFP, suggesting a conformational change in the heterotrimeric complex that results in a reduction in distance between these proteins, rather than a complete dissociation yielding free b[gamma] complexes (Bünemann et al., PNAS 26, 2003). Direct receptor stimulated interaction of the heterotrimeric G protein with the inward-rectifier subunit Kir3.4 could be demonstrated by co-expression of b2-CFP and GIRK4- YFP. Experiments are in progress to study the role of different endogenous RGS proteins on dynamics of interactions with this short pathway.

Supported by DFG and IGSN