Efremenko Anastasija V, Kryukova Elena V, Kazakov Oleg V, Ignatova Anastasia A, Shmatin Ivan I, Korabeynikova Varvara N, Toporova Victoria A, Yakimov Sergey A, Kirpichnikov Mikhail P, Nekrasova Oksana V, Feofanov Alexey V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.
Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia.
Cells. 2025 Sep 2;14(17):1364. doi: 10.3390/cells14171364.
Heterotetramerization of Kv1.1 and Kv1.2 α-subunits expands the functional diversity of voltage-gated potassium Kv1 channels in the central nervous system (CNS), thus necessitating the study of the properties of these heterochannels, including their interactions with ligands. We report on the expression, electrophysiological, and ligand-binding properties of human heterochannels Kv(1.1-1.2) formed by dimeric concatemers Kv1.1-Kv1.2 fused with fluorescent protein mKate2 in Neuro-2a cells. Kv(1.1-1.2) is a low-voltage-activated, highly active, non-inactivating channel with a fast activation rate. Its activation rate and half-maximum activation voltage are similar to that of the Kv1.1 channel, but differ from that of Kv1.2. This suggests that the membrane expression of Kv(1.1-1.2) may functionally compensate for the absence of membrane presentation of homotetrameric Kv1.1 channels in CNS. Hongotoxin 1 fused with fluorescent protein GFP (HgTx-G) is shown to be a pore-blocking ligand of Kv(1.1-1.2) with a dissociation constant of 100 pM. Using confocal microscopy and competitive binding assay, HgTx-G and cells expressing Kv(1.1-1.2), the apparent dissociation constants of the complexes between Kv(1.1-1.2) and peptides Ce1, Ce4, hongotoxin 1, MeKTx11-1, agitoxin 2, charybdotoxin, and scyllatoxin were evaluated to be 14, 33, 40, 250, 800, and >>3300 pM, respectively. Heterotetramerization of α-subunits has a different effect on the affinity of ligands compared to those for Kv1.1 and Kv1.2 channels.
Kv1.1和Kv1.2 α亚基的异源四聚化扩展了中枢神经系统（CNS）中电压门控钾离子Kv1通道的功能多样性，因此有必要研究这些异源通道的特性，包括它们与配体的相互作用。我们报道了在Neuro-2a细胞中由与荧光蛋白mKate2融合的二聚体串联体Kv1.1-Kv1.2形成的人异源通道Kv(1.1-1.2)的表达、电生理和配体结合特性。Kv(1.1-1.2)是一种低电压激活、高活性、非失活的通道，激活速率快。其激活速率和半数最大激活电压与Kv1.1通道相似，但与Kv1.2不同。这表明Kv(1.1-1.2)的膜表达可能在功能上补偿CNS中同四聚体Kv1.1通道膜呈现的缺失。与荧光蛋白GFP融合的洪戈毒素1（HgTx-G）被证明是Kv(1.1-1.2)的孔道阻断配体，解离常数为100 pM。使用共聚焦显微镜和竞争性结合测定，对Kv(1.1-1.2)与肽Ce1、Ce4、洪戈毒素1、MeKTx11-1、阿吉毒素2、蝎毒素和刺尾鱼毒素之间复合物的表观解离常数进行评估，分别为14、33、40、250、800和>>3300 pM。与Kv1.1和Kv1.2通道相比，α亚基的异源四聚化对配体亲和力有不同影响。
