LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b

Abstract

Gating of the mammalian inward rectifier Kir1.1 at the helix bundle crossing (HBC) by intracellular pH is believed to be mediated by conformational changes in the C-terminal domain (CTD). However, the exact motion of the CTD during Kir gating remains controversial. Crystal structures and single-molecule fluorescence resonance energy transfer of KirBac channels have implied a rigid body rotation and/or a contraction of the CTD as possible triggers for opening of the HBC gate. In our study, we used lanthanide-based resonance energy transfer on single-Cys dimeric constructs of the mammalian renal inward rectifier, Kir1.1b, incorporated into anionic liposomes plus PIP, to determine unambiguous, state-dependent distances between paired Cys residues on diagonally opposite subunits. Functionality and pH dependence of our proteoliposome channels were verified in separate electrophysiological experiments. The lanthanide-based resonance energy transfer distances measured in closed (pH 6) and open (pH 8) conditions indicated neither expansion nor contraction of the CTD during gating, whereas the HBC gate widened by 8.8 ± 4 Å, from 6.3 ± 2 to 15.1 ± 6 Å, during opening. These results are consistent with a Kir gating model in which rigid body rotation of the large CTD around the permeation axis is correlated with opening of the HBC hydrophobic gate, allowing permeation of a 7 Å hydrated K ion.

The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations

“…Jensen et al have carried out some extremely large simulations of gating [ 9 , 111 ]. Sansom and coworkers have used MD for several aspects of channel properties [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ,…”

“…A second line of evidence that has been interpreted in terms of the standard models comes from fluorescence quenching. FRET (fluorescence resonance energy transfer) or LRET (lanthanide resonance energy transfer—often Tb 3+ is used as the lanthanide) measurements give an indication of the distance between residues, which appears to change with applied voltage [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Again, there are difficulties with this type of measurement.…”

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP

“…Even though several publications 16,[29][30][31] point out how LRET decay curves may be evaluated it is worth to lay out the basis for the global t routine employed in our evaluation.…”

Interaction of the Motor Protein SecA and the Bacterial Protein Translocation Channel SecYEG in the Absence of ATP

“…Even though several publications 16,[29][30][31] For the donors ‫ܦ‬ , the decay is simply described by…”