Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

Abstract

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

Maturation of the functional mouse CRES amyloid from globular form

“…To address the issue of peak overlap, in addition to 2D dipoleassisted rotational resonance (DARR; 12-ms, 25-ms, and 50-ms mixing times) and NcaCX and NcoCX spectra, we acquired three-dimensional (3D) CANcoCA (Cα-N-c′-C αi-1 ), NCACX (N i -Cα i -C i,sidechain ), and NCOCX (N i -C′ i-1 -C i-1,sidechain ) spectra. The signal to noise of these spectra was improved using double exponential weighted sampling and following protocols we and others previously reported (36,37). We assigned all backbone 15 N and 13 C and most sidechain resonances for 112 residues stretching from Q32 through V142, with limited ambiguity.…”

Permeation mechanisms through the selectivity filter and the open helix bundle crossing gate of GIRK2

“…Thus, the SF tilt of GIRK2 is likely to affect the entrance of extracellular K + ions to the channel. Recently, a SSNMR study of KirBac1.1 has indicated that, the S4 binding site is expected to be involved in the conductive state of SF, and that deformation at S4 might refer to a nonconductive state [61] . Due to the homology between KirBac1.1 and Kir3.2, how the S4 binding site relates to the permeation efficiency through SF remains to be further clarified.…”

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

“…Our previous study over KirBac1.1 identified transmembrane residues with large CSPs in response to conformation exchange between the inactive and active states of the channel [65]. These residues are typically found near those known to be functional residues involved in conformational exchange within the transmembrane and extracellular regions and include both those observed in the inactive to active state transition and those found within alternate assignment pathways within the active state.…”