Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristic of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily missing a representative structure of the T1 domain is the Kv2 family. We used X-ray crystallography to solve the structure of the human Kv2.1 T1 domain. The structure is similar to other T1 domains but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer as determined with in-line SEC-MALS-SAXS and negative stain EM. The Kv2.1 T1-T1 interface involves electrostatic interactions including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. We show that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains which may reflect the versatility of Kv2.1, the only Kv subfamily that can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-EM structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic T1 domain which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the VSD/PD interface. Supported by MD simulations and electrophysiological and mutational analyses, we identify close communication between α6 helix of T1 domain, S4/S5 linker and S6T helix as responsible for the ultra-fast activation/deactivation and open state stabilisation that are unique to Kv3 channels. These findings provide fundamentally new insights into gating control and disease mechanisms and guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

The S6 gate in regulatory Kv6 subunits restricts heteromeric K+ channel stoichiometry

The Shaker-like family of voltage-gated K+ channels comprises four functionally independent gene subfamilies, Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4), each of which regulates distinct aspects of neuronal excitability. Subfamily-specific assembly of tetrameric channels is mediated by the N-terminal T1 domain and segregates Kv1–4, allowing multiple channel types to function independently in the same cell. Typical Shaker-like Kv subunits can form functional channels as homotetramers, but a group of mammalian Kv2-related genes (Kv5.1, Kv6s, Kv8s, and Kv9s) encodes subunits that have a “silent” or “regulatory” phenotype characterized by T1 self-incompatibility. These channels are unable to form homotetramers, but instead heteromerize with Kv2.1 or Kv2.2 to diversify the functional properties of these delayed rectifiers. While T1 self-incompatibility predicts that these heterotetramers could contain up to two regulatory (R) subunits, experiments show a predominance of 3:1R stoichiometry in which heteromeric channels contain a single regulatory subunit. Substitution of the self-compatible Kv2.1 T1 domain into the regulatory subunit Kv6.4 does not alter the stoichiometry of Kv2.1:Kv6.4 heteromers. Here, to identify other channel structures that might be responsible for favoring the 3:1R stoichiometry, we compare the sequences of mammalian regulatory subunits to independently evolved regulatory subunits from cnidarians. The most widespread feature of regulatory subunits is the presence of atypical substitutions in the highly conserved consensus sequence of the intracellular S6 activation gate of the pore. We show that two amino acid substitutions in the S6 gate of the regulatory subunit Kv6.4 restrict the functional stoichiometry of Kv2.1:Kv6.4 to 3:1R by limiting the formation and function of 2:2R heteromers. We propose a two-step model for the evolution of the asymmetric 3:1R stoichiometry, which begins with evolution of self-incompatibility to establish the regulatory phenotype, followed by drift of the activation gate consensus sequence under relaxed selection to limit stoichiometry to 3:1R.

Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

The activity of voltage-gated K+ (KV) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH2-terminal tetramerization domain (T1) of KV channels is important for proper channel assembly, association with regulatory KV β-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.