The Lipid Activation Mechanism of a Transmembrane Potassium Channel

Abstract

Membrane proteins and lipids coevolved to yield unique coregulatory mechanisms. Inward-rectifier K⁺ (Kir) channels are often activated by anionic lipids endemic to their native membranes and require accessible water along their K⁺ conductance pathway. To better understand Kir channel activation, we target multiple mutants of the Kir channel KirBac1.1 via solid-state nuclear magnetic resonance (SSNMR) spectroscopy, potassium efflux assays, and Förster resonance energy transfer (FRET) measurements. In the I131C stability mutant (SM), we observe an open-active channel in the presence of anionic lipids with greater activity upon addition of cardiolipin (CL). The introduction of three R to Q mutations (R49/151/153Q (triple Q mutant, TQ)) renders the protein inactive within the same activating lipid environment. Our SSNMR experiments reveal a stark reduction of lipid-protein interactions in the TQ mutant explaining the dramatic loss of channel activity. Water-edited SSNMR experiments further determined the TQ mutant possesses greater overall solvent exposure in comparison to wild-type but with reduced water accessibility along the ion conduction pathway, consistent with the closed state of the channel. These experiments also suggest water is proximal to the selectivity filter of KirBac1.1 in the open-activated state but that it may not directly enter the selectivity filter. Our findings suggest lipid binding initiates a concerted rotation of the cytoplasmic domain subunits, which is stabilized by multiple intersubunit salt bridges. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K⁺ conduction pathway. This work highlights universal membrane protein motifs, including lipid-protein interactions, domain rearrangement, and water-mediated diffusion mechanisms.

Figure 1.

Structural similarities of lipid-activated Kir channels to KirBac1.1. Surface charge maps of KirBac1.1 (a) (PDB ID: 2wll) and Kir2.2-PIP complex (b) (PDB ID: 3spi) are depicted with red denoting anionic residues and blue specifying cationic residues. The cationic sites thought to bind anionic lipids are boxed in magenta in each structure. These binding pockets are expanded in (c) and (d). Key cationic residues are labeled and shown in blue and gray. Cocrystallized lipid head groups are illustrated in each. The choline headgroup in 2wll is depicted in orange, gray, and blue in (c), and the PIP₂ analog is shown in red, green, and orange in (d). Distances are provided between R49/151/153 Cζ and the cocrystallized phosphocholine group. (e) Sequence alignment of KirBac1.1 and 3.1 along with various human Kir channels. Homologous regions are boxed in blue with residues in the same group shown in red. The highly conserved c-linker motif (R—P—K—K—R) is highlighted in purple.

Figure 2.

K⁺ efflux assays and FRET analysis of SM and mutant channels. (a) K⁺ efflux assays of SM–KirBac1.1 and two inactivating mutants R151/153Q (DQ) and TQ reconstituted into POPE/POPG (75:25) (w/w). (b) K⁺ efflux assays of the same proteins, performed in POPE/POPG/CL (75:24:1) (w/w) bilayers. The protein free (PF) negative controls for each lipid condition are depicted in black. Decay rates (±RMS) (a, inset) are negligible for both mutants with the exception of DQ in 1% CL. (c) FRET buildup curves after the addition of proteinase K. Curves are for both DQ and TQ mutants of the SM–G249C (GC) FRET construct in POPE/POPG bilayers with or without 5% (w/w) PIP₂. (d) FRET efficiency values with significant reduction for GC in the presence of PIP₂, and negligible differences for each mutant in the presence of PIP₂.

Figure 3.

Spectral evidence for arginine–lipid interactions. (a) 2D NcoCX spectra of SM KirBac1.1 in black and TQ overlaid in red. Both samples are reconstituted into a 75:20:5 POPE/POPG/CL (PE/PG/CL) lipid mixture. Boxed in blue are arginine Cζ outliers at ~156.9 ppm, which are observed in SM but absent in TQ. (b) ¹³C–³¹P REDOR dephasing experiment for SM targeting aforementioned Cζ outliers (gray box) with an inset of the proposed interaction with lipid phosphate with the control (S₀) in black and dephased spectra (S) in red. Percentage (S/S₀) of dephased indicated in red for the “bulk” and outlier arginine peaks. Residuals between S₀ and S in blue. (c–e) NA-¹³C-lipid rINEPT temperature series of SM (c), TQ (d), and lipids only (e) with first derivative (ξ) insets of the respective melting curves. Error was determined via RMS of the spectra (~3%) and is within the size of the symbols denoting each point.

Figure 4.

Notable differences in standard ¹³C–¹³C DARR spectra for the U-¹³C,¹⁵N TQ(red) and SM (black) KirBac1.1 samples in the standard PE/PG/CL mixture. Residues A18/154 (left) undergo CSPs in TQ; T120 (middle) experiences a minor CSP with a stark reduction in polarization, and in I138 (right), the Cα-Cδ is only present in the SM. Full spectra are provided in Figure S6.

Figure 5.

Spectral evidence of the increased water accessibility of the TQ mutant. (a–c) Water-edited spectra of SM and TQ samples in black and red, respectively. Charged residue populations Glu/Asp Cδ/Cγ that we have assigned (a) (Figure S1) and Arg Cζ populations that appear much stronger in TQ over SM in both the ¹H–¹³C HETCOR spectra (a, b) and Glu Cδ. (d) Close-up of CTD charged residues in a SWISS-MODEL of the 1p7b structure with missing residues 196–205 and 295–300 modeled in. (e, f) Buildup curves for both SM and TQ in black and red, respectively. The greater overall exposed backbone and charged residue surface were observed for TQ; the carboxyl groups in particular (f) show a dramatic increase. Lines are best-fits based upon eq 11. Error bars are determined via RMS of spectral noise.

Figure 6.

Site specific solvent accessibility of KirBac1.1. (a) Water accessibility map of KirBac1.1 generated from assignments of water-edited 2D DARR experiments with 5 ms of ¹H–¹H mixing (b–e) of both SM (black) and TQ (red) samples in our standard PE/PG/CL mixture (Figures S16 and S17). Sites and assignments are color coded, corresponding to those that appear more strongly in SM (blue) or more strongly in TQ (red) or are CSPs from SM to TQ (orange). (b, c) Distinct differences observed for select proline and isoleucine side chain correlations. (d) Numerous charged residues found primarily in the C-terminus were more water exposed in TQ. (e) Glycine C′-Cα correlations for selectivity filter and TM2 residues. Green arrows indicate CSPs of G137C′-Cα, G114C′-Cα, and I138Cγ1-Cδ. (f) Residues not represented in the 1p7b crystal structure with those more exposed in TQ or SM in red and blue, respectively. Residues 310 to 333 do not appear in the crystal structure and remain uncharacterized.

Figure 7.

Cartoon of the proposed structural changes to SM KirBac1.1 (left), upon removal of the anionic lipid binding site in the TQ mutant (right). Membrane and aqueous regions are highlighted in yellow and blue, respectively. Solvent exposed protein surface for each respective state is highlighted in light blue, and potassium ions are depicted as yellow circles. The bottom-up perspective (bottom) of CTD motions is shown for both SM and TQ.