Gating Motions of KirBac1.1 Cytoplasmic Domain with Respect to Transmembrane Domain Revealed by FRET

Abstract

KirBac1.1 is a bacterial inward rectifier potassium (Kir) channel, which, contrary to its eukaryotic homologues, is strongly inhibited by phosphatidylinositol-4,5-bisphosphate (PIP2). The most recent crystal structures of eukaryotic Kir2.2 in complex with PIP2 indicate that the TM-CD linker forms a short a-helix in the presence of PIP2. As a result, the cytoplasmic domain of Kir2.2 moves about 6 angstrom towards the membrane surface. However, the ‘KKR’ motif in the TM-CD linker of Kir2.2, which directly interacts with PIP2, is absent in KirBac1.1 and the question arises: how does KirBac1.1 cytoplasmic domain move in response to PIP2 inhibition? In the present work, we have made KirBac1.1 tandem tetramer constructs and purified KirBac1.1 tandem proteins successfully. Reconstituted liposome flux assays indicate that the KirBac1.1 tandem protein remains functional, and retains sensitivity to PIP2 inhibition. We introduced two cysteine into the KirBac1.1 tandem tetramer, one at the extracellular loop of subunit 1 (G85 or T120) and one in the cytoplasmic domain of subunit 2 (A273). We labeled these cysteines with a FRET dye pair (Alexa-Fluor 488 and DABCYL) and measured FRET efficiencies in protein samples reconstituted into liposomes, in the absence and presence of PIP2. Our results indicate that the KirBac1.1 cytoplasmic domain moves ∼2-3 angstrom away from the transmembrane domain in the presence of PIP2 - opposite the direction suggested from eukaryotic Kir2.2 crystal structures in the presence and absence of PIP2. Reversed PIP2-dependent motions of the cytoplasmic domain with respect to the transmembrane domain between prokaryotic and eukaryotic Kir channels may explain their differential response to PIP2 modulation.