Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel

G-protein-gated inward rectifying potassium channels (GIRKs) require Gβγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by Gβγ and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to Gβγ-binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions.

7.6 at room temperature). After the solution was passed through the column, 15 mL of wash buffer consisting of buffer A supplemented with an additional 0.5% DDM was applied. The column was then exchanged into several column volumes of buffer A or until a steady baseline for 280 nm absorbance was established. The protein was eluted with 40 mL of buffer B (150 mM KCl, 30 mM Tris, 300 mM imidazole, 10% glycerol and 0.022% DDM, pH 7.6 at room temperature). Peak fractions were pooled, then loaded onto a HiPrep 26/10 desalting column (GE Healthcare) pre-equilibrated with Buffer C (150 mM KCl, 30 mM Tris, 10 mM βmercaptoethanol (BME), 10% Glycerol and 0.022% DDM, pH 7.5 at room temperature). Peak fractions were pooled and then loaded onto two 5-mL StrepTrap HP Columns (GE Healthcare) connected in tandem preequilibrated in buffer C. Buffer D (150 mM KCl, 30 mM Tris, 10 mM BME, 4 mM d-desthiobiotin, 10% glycerol and 0.022% DDM, pH 7.5 at room temperature) was applied to elute recombinant protein from the StrepTrap HP Columns. Peak fractions were pooled and once again loaded onto a HiPrep 26/10 desalting column preequilibrated with Buffer C to remove d-desthiobiotin. Peak fractions were pooled before adding His-tagged TEV protease produced in-house 2 . The mixture was incubated overnight at 9 ºC and then filtered through a 0.45 μm syringe filter (Pall Corporations). The protein solution was loaded onto a 5-mL HisTrap HP column (GE Healthcare) equilibrated in buffer E (150 mM KCl, 30 mM Tris, 25 mM imidazole, 10% glycerol and 0.022% DDM, pH 7.6 at room temperature). Flow-through containing the untagged GIRK2 was collected and concentrated using a 100,000 MWCO concentrator (Millipore) to roughly 2 mg/ml as determined by UV absorbance (with coefficient of 1 Abs = 1 mg/mL). The protein was flash-frozen in liquid nitrogen, stored at -80 ºC, and typically used within two weeks. These steps, including the desalting step following the StrepTrap, are crucial for the stability of the protein prior to the final polishing step for mass spectrometry analysis.

GIRK2 Polishing for Native Mass Spectrometry Studies
About ~1 mg of purified GIRK2 protein solution was thawed and added to buffer F (150 mM KCl, 50 mM NaCl, 30 mM Tris, 10 mM BME, 1 mM DTT [1,4-dithiothreitol], 10% glycerol, 15 mM DHPC [1,2-diheptanoyl-snglycero-3-phosphocholine] and 0.022% DDM, pH 7.5 at room temperature). The ratio of protein solution to buffer F added is 1:8 by volume and the solutions were kept at 15 ºC before mixing. The mixture was immediately concentrated in a 100,000 MWCO concentrator at 3,000 g and 15 ºC for 3 minutes, and the mixture was manually re-suspended with a pipette. This process was repeated until 1 mL of solution remain to avoid precipitation of the protein. 10 mL of buffer C was added to the concentrator and the sample slowly concentrated to a final volume of 500 L with repeated resuspension of the solution in the concentrator every 3 minutes to minimize aggregation. The mixture was then filtered through a 0.22 μm spin-filter (Millipore) before being injected into a Superdex 200GL 10/300 (GE Healthcare) column equilibrated in buffer G (150 mM KCl, 50 mM NaCl, 30 mM Tris, 10% glycerol, 0.07% C10E5 (decylpentaglycol, Anatrace), and pH 7.4 at room temperature). Peak fractions containing delipidated GIRK2 were pooled and concentrated in a 100,000 MWCO concentrator until 50 L total volume was reached. Concentrated proteins were either flash-frozen in liquid nitrogen and stored at -80 ºC, or used directly by exchanging into MS buffer (100 mM ammonium formate, 0.065% C10E5, and pH 7.2 at room temperature) using a centrifugal buffer exchange device (MicroBio-Spin6, Bio-Rad) following manufacture's protocol. Ammonium formate is used instead of the typical ammonium acetate due to GIRK2 having slightly better shelf-life in this buffer. For convenience, the sample in MS buffer was aliquoted into 2 L fractions, flash-frozen in liquid nitrogen and stored at -80 ºC until used for experiments. Notably, we found no observable difference in mass-spectral quality after a single freezethaw of GIRK2 proteins up until this point of our purification regime.

Preparation of Phospholipids and Other Ligands for MS Binding Studies
Phospholipid stock solutions were prepared as previously described. 3 Briefly, chloroform was removed from the phospholipid ampoules, which were pre-aliquoted by Avanti Polar Lipid, by gentle nitrogen gas flow and then by desiccation overnight. For lipids purchased in powder form, chloroform was added, and the solution was transferred to a new glass vial and dried as described above. The dried lipid films were solubilized in the MS buffer. Lipid concentrations were calculated directly from the weight of each aliquots in individual vials.
The molar ratio of wild-type or R201A GIRK2 to phospholipids was held constant at a ratio of 1:6. Protein aliquots were kept on dry ice until mixing with lipid solution at a 1:1 volume ratio. Specifically, GIRK2 and lipid mixtures were held at a final concentration of about 500 nM and 3 μM, respectively. The R201A GIRK2 and lipid mixtures were at a final concentration of about 825 nM and 5 μM, respectively. These protein-lipid mixtures were allowed to incubate for 2 minutes at room temperature before loading into a gold-coated glass capillary tip produced in-house as previously described. 4 We have found that longer incubation time does not change the mole fractions of apo and lipid-bound proteins. These findings are in agreement with our previous studies for other membrane proteins. 3 Ivermectin (Alfa Aesar) was solubilized in DMSO and/or 100% ethanol, and dilutions made in MS buffer. No noticeable precipitation of ivermectin was observed after dilution in MS buffer until a final concentration of 2mM in 20% DMSO or 5% ethanol, 3% DMSO is reached. This sample was mixed with GIRK2 in a 1:5 ratio by volume of ivermectin to GIRK2. Tertiapin Q (TPNQ, Alomone Labs) was diluted directly in MS buffer to 100 μM or lower, and mixed with GIRK2 in a 1:1 ratio by volume.

Native Mass Spectrometry and Data Analysis
A Synapt G1 HDMS instrument (Waters Corporation) with a 32k RF generator was used for most of the data collected. Instrument parameters were tuned to maximize ion intensity and simultaneously preserve the native-like state of GIRK2. The capillary voltage was set to1.75 kV, sampling cone voltage at 200 V, extractor cone voltage at 10 V and argon flow rate at 7 mL/min (5.2 x 10 -2 mbar). The T-wave settings for trap (300 ms and 1/1.0 V), IMS (300 ms and 1/18 V) and transfer (100 ms and 1/10 V), source temperature (90 ºC) and trap bias (35 V) were also optimized. Trap and Transfer Collision Voltage were set to 100 V and 60 V, respectively, unless otherwise noted. Ion mobility mass spectrometry data were processed using the software program Pulsar 5 and de-convoluted using UniDec 6 followed by converting intensities of GIRK2 and PIP-GIRK2 species to mole fractions as described previously. 3 High-resolution MS spectra were collected using an Exactive Plus with extended mass range (EMR) from Thermo Scientific. Samples were analyzed using both the original instrument and a modified reverse entry ion source (REIS) coupled to the HCD cell of the Orbitrap. 7 Gold-coated capillaries described above were loaded with sample. Operating conditions of the REIS were described in detail previously; briefly, ions are generated by nano-ESI, focused with a RF ion funnel, and transferred to the HCD cell by an octupole ion guide. All mass spectra were collected with 10 µscans at 17 500 mass resolution with an ion injection time of 200 ms. Collision energies of 20 eV in-source CID and 70 HCD collision energy for normal operating conditions and 110 HCD collision energy for REIS. These energies were chosen to effectively desolvate and strip detergents while minimizing perturbations to protein-lipid and subunit interactions. Native MS data from the Exactive Plus EMR is analyzed and mass species assigned using UniDec. 6

Top-down and Bottom-up Mass Spectrometry
A sample of denatured GIRK2 was prepared using method described by Campuzano and co-workers 8 with minor modifications. A TSKgel Phenyl-5PW RP column (7.5 cm x 4.6 mm, Tosoh Bioscience) was connected to an AKTA Avant (GE Healthcare) an equilibrated in 30% n-propanol, 0.1% Formic Acid (FA), and 0.1% Trifluoracetic acid (TFA). An aliquot of GIRK2 with purification tags removed by TEV protease was incubated with 5 mM BME prior to injection onto the column. The denatured GIRK2 protein was eluted with a gradient over three mL to 100% n-propanol, 0.1% FA, and 0.1% TFA at a flow rate of 1 mL/min. The peak fraction containing denatured GIRK2 was directly infused into the front-end source of Exactive Plus EMR instruments with the following settings: Scan resolution was set to 17 500, 100 eV in-source CID and 10 HCD collision energy was used, as well as 125 degrees ºC for capillary temperature. For bottom-up analysis, TEVprocessed GIRK2 was digested with trypsin overnight, then applied through a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system, consisting of a Dionex Ultimate Fusion mass spectrometer. LC system solvents were water + 0.1% FA (A) and acetonitrile with 0.1% FA (B). Tryptic peptides were eluted over 60 minute gradient from 2% B from 0 to 5 min, 2% to 45% from 5 to 37 min, 45% to 90% from 38 to 46 min, and down to 2% from 46 to 60 min at a flow rate of 0.4 uL/min. The mass spectrometer ion source was set to have a spray voltage of 2.3 kV, ion transfer tube temperature of 275 ºC, the scan range was m/z 400-1600 with a resolution of 120,000. MS/MS acquisition was performed with 3 s cycle time. The intensity threshold was set to 5000, Ions with charge states 1+ to 6+ were sequentially fragmented by high energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28%. The dynamic exclusion duration was set as 60 s. Raw files were analyzed using the Thermo Proteome Discoverer (v2.1.0.81) software platform. The mass spectrometry data was analyzed using SEQUEST with the following parameters: the protein sequence database contained only the recombinant GIRK2 sequence, trypsin selected as the enzyme, dynamic (or variable) modifications included protein N-terminal acetylation, oxidation, serine, threonine and tyrosine phosphorylation. Carbamidomethylation of cysteine was set as a fixed modification since trypsin digested samples was treated with iodoacetamide. Mass tolerances for precursor ions was set to 10 ppm, and fragment ions set to Δ0.6 Da. Limits for peptide length searched range from 6 to 144, maximum delta Cn is set to 0.05. Maximum number of allowed missed cleavages is 2.

Supplementary Tables
Supplementary Table 1. Calculated and measured masses for GIRK2 R201A and GIRK2 bound to different ligands acquired on a Synapt G1 instrument. Measured mass was determined by deconvolution of native mass spectra using the program, UniDec. 6 For ligands bound to GIRK2, the addition of mass to the apo GIRK2 mass is reported. The reported standard deviation was calculated directly from the width of the zerocharge mass spectrum from UniDec after deconvolution.