• Le 19 octobre 2017
    Institut de Recherche en Santé - 8 quai Moncousu - Nantes
    Amphithéâtre Denis Escande
     
  • 15h30 - 17h00

Importance des sous-unités alpha silencieuses de canaux ioniques

Silent Kv6.4 subunits assemble with Kv2.1 into heterotetramers with a 2:2 stoichiometry wherein Kv6.4 subunits are positioned diagonally.

Alain Labro, Université d'Anvers, Belgique


Abstract

Members of the voltage-gated K+ (Kv) subfamilies Kv5, Kv6, Kv8 and Kv9, which are collectively identified as electrically silent (KvS) subunits, selectively modulate the biophysical properties of Kv2 channels by forming heterotetrameric Kv2/KvS channels. However, the stoichiometry of only Kv2.1/Kv9.3 channels has been investigated proposing a composition of three Kv2.1 and one Kv9.3 subunit (3:1 ration) based on data from cell fluorescence resonance energy transfer (FRET) experiments. Despite further investigation, it is assumed that also the other Kv2/KvS channels assemble with a 3:1 ratio. In this study the stoichiometry of Kv2.1/Kv6.4 channels was determined using single molecule step-wise bleaching experiments, which is a more accurate approach to determine the number of labelled subunits within channels compared to previously used FRET techniques. Furthermore, this approach allows to determine the stoichiometry of only functionally folded channels expressed at the cell surface. Labelling Kv6.4 with GFP, the fluorescence of heteromeric channels is bleached in 2-steps which indicates that the functional channels are composed of two silent Kv6.4 and two Kv2.1 subunits, thus Kv2.1/Kv6.4 channels assemble with a 2:2 ratio. To investigate this further and to determine the composition of Kv2.1 and Kv6.4 subunits within these 2:2 ratio heterotetramers, concatemeric constructs were created. Kv2.1-Kv6.4 (2_6) or Kv6.4-Kv2.1 (6_2) dimers displayed the characteristic inactivation properties of Kv2.1/Kv6.4 channels obtained from co-transfecting the individual subunits. Analysis of tetrameric constructs revealed that the position of the Kv6.4 subunits was crucial. No current expression was detect from the tetrameric construct in which two Kv6.4 subunits were positioned side by side (2_6_6_2), suggesting that the Kv6.4 subunit has no compatible interaction sites with itself. In contrast, the tetramer with an alternating subunit arrangement (2_6_2_6) yielded functional channels that behaved as Kv2.1/Kv6.4 channels. This indicated that the Kv6.4 subunits need to be positioned diagonally within the tetramer. Additionally, tetramers that represented a 3:1 stoichiometry (2_6_2_2) also displayed the Kv6.4-characteristic inactivation behavior. Taken together, this data indicates that besides the generally assumed 3:1 configuration the Kv2.1:Kv6.4 heterotetramers assemble with a 2:2 ratio, wherein the silent Kv6.4 and Kv2.1 subunit need to be positioned diagonally.

 

Trafficking and heteromerization of two-pore domain potassium (K2P) channels.

Delphine Bichet, Université de Nice Sophia Antipolis

Abstract

Among K2P channels, some of them turned out to be difficult to express in heterologous systems and were coined “silent subunits”. Our studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For these channels, silence is related to a unique combination of intracellular retention and low intrinsic activity. These intracellular localizations are due to trafficking signals located in the cytoplasmic parts of the channels. When these motives are mutated or masked, channels are redistributed at the plasma membrane and produce some measurable currents. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins.

Nearly 80 human genes encode potassium channels and this diversity is further increased by post-transcriptional and post-translational mechanisms. For these multimeric channels, heteromerization may also raise the number of channels to hundreds. Our studies show that mixing and matching different K2P subunits generate channels with novel properties. Heterodimeric combinations have properties different from those of the corresponding homodimers, including subcellular localization, single channel behavior, regulations by kinases and sensitivity to pharmacological agents. This implies that any excitable cell can adjust its response by simply modulating the ratio of expressed K2P subunits and that heteromerization has to be considered when analyzing in vivo functions of these channels but also when screening new potential therapeutic drugs.