Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators

Abstract Kv3 ion-channels constitute a class of functionally distinct voltage-gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 Å resolution using full-length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well-resolved Zinc site and displaying a rotation by 35°. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage-gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

Patch-clamp recordings were performed using the automated recording system QPatch-16x/QPatchII-48x (Sophion Bioscience, Denmark). HEK-293 cells stably expressing human flWT-Kv3.1b was used for the experiments. On the day of the experiment the cells were detached by Detachin and resuspended in serum free medium containing 25 mM HEPES and 100U/ml Penicillin/Streptomycin. For each experiment cells were centrifuged, media removed and the cells were resuspended in extracellular buffer containing (in mM): 145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES and 10 glucose; pH 7.4 adjusted with NaOH, 305 mOsm. Single cell whole-cell recordings were carried out using an intracellular solution containing (in mM): 120 KCl, 32.25/10 KOH/EGTA, 5.374 CaCl 2 , 1.75 MgCl 2 , 10 HEPES, 4 Na 2 ATP, pH 7.2 adjusted with KOH, 295 mOsm. Cell membrane potentials were held at -80 mV and currents were evoked by voltage steps (50 ms duration) from -70 mV to +10 mV (in 10 mV increments). Vehicle (0.33% DMSO) or increasing concentration of compound were applied and the voltage protocol was run 3 times/concentration (resulting in 3 min cpd incubation time). Five increasing concentrations of compound were applied to each cell. Leak subtraction protocol was applied at -33% of the sweep amplitude, and serial resistance values were constantly monitored. Any cell where serial resistance exceeded 25 MOhm, membrane resistance less than 200 MOhm or current size at -10 mV less than 200 pA was eliminated from the subsequent analysis.
Data analysis was performed using Sophion's QPatch analyzer software in combination with Microsoft Excel™ (Redmond, WA,USA). Current voltage relationships were plotted from the peak current at the individual voltage steps normalized to the vehicle addition at 10 mV. The voltage threshold for channel activation was defined as 5% activation of the peak current at 10 mV in presence of vehicle. The activity of the compounds was described as the ability to shift this current voltage relationship to more hyperpolarized potentials and is given as the maximum absolute shift possible at the tested concentrations (0.37, 1.11, 3.33, 10, 30 µM). Concentration response curves were plotted from the threshold shift at the individual concentrations and were fitted excel fit model 205 sigmodal dose-response model (fit=A+((B-A)/1+((C/x)^D)))), where A is the minimum value, B the maximum value, C the EC50 value and D the slope of the curve. The concentration needed to shift the threshold 5 mV was readout from this curve (EC Δ5mV ).
A. After affinity purification the protein sample was subjected to superose 6 Size Exclusion chromatography. The peak corresponding to the tetramer is indicated with an arrow. B. The final sample was analyzed by SDS-PAGE. Positions of the protein markers bands are indicated with arrows on the left side. C. The final protein sample was analyzed by negative staining. Graphical description of the processing scheme applied to the apo flWT-Kv3.1a dataset. Full description can be found in the "Materials and Methods" section.

B
A C

Cartoon representation of the cylindrical radius within the pore
The pore radius is shown as calculated with the HOLE2 program, indicating an open channel at the lower gate region indicated with a red arrow.

Figure S6
Characterization of Lu AG00563 on flWT-Kv3.1b channel A. Representative traces for HEK293 cells stably expressing flWT-Kv3.1b as measured by automated electrophysiology using QPatch. Cells were subjected to a 50 ms step to -10 mV followed by 100 ms recovery before an IV protocol was applied from -70 mV to 10 mV in 10 mV increments ( Graphical description of the processing scheme applied to the flWT-Kv3.1a in presence of Lu AG00563 dataset. Full description can be found in the "Materials and Methods" section.  Region of disease related mutations in S4 in the VSD that were reported in the Kv3 class. A. Sequence alignment of human Kv3.1, Kv3.2 and Kv3.3, showing a high degree of conservation in the sequence of S4 in the VSD. Reported disease related mutations occurring in a class member are shown in bold. The corresponding residue in another class member is indicated B. Representation of the disease relevant Arg317, Arg320, and Thr325 residues (purple) in S4 within the VSD (light green), in the vicinity of the Lu AG00563 ligand pocket.