Structural insights into the function, dysfunction and modulation of Kv3 channels

Fig. 1.. Cryo-EM apo structure of hKv3.1a in an open conformation. A) Lateral and outside views of the hKv3.1a tetrameric assembly (PDB ID: 7PHI) depicting the transmembrane domain (TMD) encompassing the VSD and PD, and the cytoplasmic T1 domain (cT1D). The top view demonstrates the domain-swapped architecture of the tetramer, a feature that is characteristic of Kv1, Kv2, Kv3, Kv4 and Kv7 channels. B) Single subunit overlay of the hKv3.1a and Kv1.3 structures. While there is an excellent agreement between the transmembrane domains of the two structures, the conformations of their cT1Ds differ substantially. Relative to each other, a bottom view reveals a 48° rotation. Also, the α–6 helix of Kv3.1 is rotated 90° inward, relative to the position of the equivalent segment in Kv1.3. C) Zoomed in view of the cT1D α–6 helix, which together with the S4-S5 linker and the distal segment of the S6 helix forms the gating triad that controls fast voltage-dependent gating. These visualizations were created in PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC).

Fig. 2.. The Kv3.1-C78Y and Kv3.1-C78W exhibit a de novo Π-Π stacking interaction in the cytoplasmic T1 domain. A) Structural model of the Kv3.1-C78Y variant. This model was created as previously described (Clatot et al., 2024). The colored side chains of Y78 (orange) and Y109 (cyan) indicate the location of the Π-Π stacking interaction within the cT1D. As a reference, the blue spheres represent bound Zn++. B) Zoomed-in view of the Π-Π stacking interaction between Y78 and Y09 of the α–6 helix. C) Zoomed-in view of the Π-Π stacking interaction between W78 and Y109 of the α–6 helix. The model of Kv3.1-C78W was created using AlphaFold3 (Jumper et al., 2021).

Fig. 3.. The cryo-EM structure of the AUT5-bound conformation of hKv3.1a. A) Outside view (left) of hKv3.1a depicting four equivalent AUT5 binding pockets. AUT5 occupies the inter-subunit interfaces between the VSD and the PD. The extracellular turrets mark the location of the binding sites. The lateral view (right) shows that AUT5 occupies a relatively deep pocket that is capped by the turret. B) Zoomed in view of the AUT5 binding site. AUT5 pocket is lined by the S4 helix on the VSD side and the S5 helix on the PD side. In the bound conformation, the turrets fold over the occupied site and their C-terminal sides adopt a two-turn α-helix structure. Part of the pocket is also exposed to the lipid bilayer. Using the coordinates of the AUT5 bound structure of hKv3.1a at a resolution of 2.9 Å (PDB ID: 7PHI), these visualizations were created in PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC).

Fig. 4.. The Kv3.4 NTID faces a steric clash at the lateral intracellular windows of the Kv3.4 channel. A) Kv3.4 model highlighting the lateral opening (cyan) that the NTID must cross to reach the intracellular mouth of the pore. This model does not include the free NTIDs. B) The NMR structure of the Kv3.4 NTID displayed in two orientations (PDB ID: 1ZTN). For improved visualization of phosphorylation sites (highlighted in purple), these structures are displayed enlarged relative to the image of the Kv3.4 model on panel A. C) Overlay of the Kv3.4 model plus the appropriately scaled View 2 of the NTID. This image shows that the lateral opening is not large enough to allow the NTID to enter the internal vestibule of the Kv3.4 channel and reach its internal mouth. The Kv3.4 model was created using AlphaFold3 (Jumper et al., 2021) and the visualizations were created using PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC).

Fig. 5.. Working model of synaptic plasticity governed by phosphorylation of the Kv3.4 channel in dorsal horn synapses of the spinal cord. A nociceptive stimulus may cause repetitive DRG neuron spiking at frequencies that induce cumulative Kv3.4 inactivation and UDSB (left). Consequently, the dorsal horn (DH) glutamatergic synapse that receives this input in laminae I and II undergoes activity-dependent facilitation to relay a strong pro-nociceptive signal. To regulate this signal, released glutamate may also act on presynaptic mGluR5 to trigger a Gq/11-dependent signaling cascade that activates PKC, which would then phosphorylate the Kv3.4 NTID. This phosphorylation destabilizes Kv3.4 inactivation, shortens the AP and opposes UDSB. As a result, the modulated synapse is now depressed and nociceptive signaling is dampened. Glutamatergic gating interneurons in the DH could similarly activate presynaptic mGluR5 to modulate nociceptive signaling.