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

ABSTRACT: 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.

SUBMITTER: Pisupati A

PROVIDER: S-EPMC6279357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: In-frame deletion mutation (Del-L955) in NaV1.7 sodium channel from a kindred with erythromelalgia hyperpolarizes activation. RESULTS: Del-L955 twists the S6 helix, displacing the Phe960 activation gate. Replacement of Phe960 at the correct helical position depolarizes activation. CONCLUSION: Radial tuning of the activation gate is critical to the activation of NaV1.7 channel. SIGNIFICANCE: Structural modeling guided electrophysiology reveals the functional importance of radial tuning of the S6 segment. Voltage-gated sodium (NaV) channels are membrane proteins that consist of 24 transmembrane segments organized into four homologous domains and are essential for action potential generation and propagation. Although the S6 helices of NaV channels line the ion-conducting pore and participate in channel activation, their functional architecture is incompletely understood. Our recent studies show that a naturally occurring in-frame deletion mutation (Del-L955) of NaV1.7 channel, identified in individuals with a severe inherited pain syndrome (inherited erythromelalgia) causes a substantial hyperpolarizing shift of channel activation. Here we took advantage of this deletion mutation to understand the role of the S6 helix in the channel activation. Based on the recently published structure of a bacterial NaV channel (NaVAb), we modeled the WT and Del-L955 channel. Our structural model showed that Del-L955 twists the DII/S6 helix, shifting location and radial orientation of the activation gate residue (Phe(960)). Hypothesizing that these structural changes produce the shift of channel activation of Del-L955 channels, we restored a phenylalanine in wild-type orientation by mutating Ser(961) (Del-L955/S961F), correcting activation by ?10 mV. Correction of the displaced Phe(960) (F960S) together with introduction of the rescuing activation gate residue (S961F) produced an additional ?6-mV restoration of activation of the mutant channel. A simple point mutation in the absence of a twist (L955A) did not produce a radial shift and did not hyperpolarize activation. Our results demonstrate the functional importance of radial tuning of the sodium channel S6 helix for the channel activation.

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

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

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