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Determining the Correct Stoichiometry of Kv2.1/Kv6.4 Heterotetramers, Functional in Multiple Stoichiometrical Configurations

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Determining the Correct Stoichiometry of Kv2.1/Kv6.4 Heterotetramers, Functional in Multiple Stoichiometrical Configurations Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations Lena Möllera,1, Glenn Regnierb,1, Alain J. Labrob, Rikard Bluncka,c,2, and Dirk J. Snydersb,2 aDepartment of Biochemistry, Université de Montréal, Montréal, QC, Canada H3C 3J7; bLaboratory for Molecular, Cellular and Network Excitability, Department of Biomedical Sciences, University of Antwerp, 2000 Antwerp, Belgium; and cDepartment of Physics, Université de Montréal, Montréal, QC, Canada H3C 3J7 Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved March 5, 2020 (received for review September 17, 2019) The electrically silent (KvS) members of the voltage-gated potas- controlling tetramerization of compatible subunits (14, 15). To sium (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9 selectively modulate tune the native Kv currents to tissue-specific requirements, each Kv2 subunits by forming heterotetrameric Kv2/KvS channels. tissue expresses a characteristic set of α-subunits, which are divided Based on the reported 3:1 stoichiometry of Kv2.1/Kv9.3 channels, into several subfamilies based on sequence homology. In the case of we tested the hypothesis that Kv2.1/Kv6.4 channels express, in the Shaker-related subunits, eight different subfamilies can be dis- contrast to the assumed 3:1, in a 2:2 stoichiometry. We investigate tinguished: Kv1–Kv6 and Kv8–Kv9 (16). Within each of the the Kv2.1/Kv6.4 stoichiometry using single subunit counting and Kv1–Kv4 subfamilies, subunits cannot only oligomerize into functional characterization of tetrameric concatemers. For selecting homotetramers, but also into heterotetramers, increasing the di- the most probable stoichiometry, we introduce a model-selection versity of Kv channel complexes. Members of the Kv5, Kv6, Kv8, method that is applicable for any multimeric complex by investigat- and Kv9 subfamilies, on the other hand, are unable to form func- ing the stoichiometry of Kv2.1/Kv6.4 channels. Weighted likelihood tional channels, even though they have the typical topology of a Kv calculations bring rigor to a powerful technique. Using the weighted- α-subunit and are therefore designated silent Kv (KvS) subunits likelihood model-selection method and analysis of electrophysiolog- (17). However, they do selectively interact with members of the Kv2 ical data, we show that Kv2.1/Kv6.4 channels express, in contrast to subfamily, forming functional Kv2/KvS heterotetramers that pos- BIOPHYSICS AND COMPUTATIONAL BIOLOGY the assumed 3:1, in a 2:2 stoichiometry. Within this stoichiometry, the sess unique biophysical and pharmacological properties. Generally, Kv6.4 subunits have to be positioned alternating with Kv2.1 to ex- they slow the activation and deactivation kinetics, shift the voltage press functional channels. The variability in Kv2/KvS assembly in- dependency of activation and inactivation, and reduce in heterol- creases the diversity of heterotetrameric configurations and ogous expression systems the current density relative to Kv2 extends the regulatory possibilities of KvS by allowing the presence homotetramers. Therefore, these KvS subunits are considered of more than one silent subunit. modulatory α-subunits of the Kv2 subfamily. Oligomerization of Kv subunits into a functional channel is Kv channels | single subunit counting | silent subunits | model selection thought not to occur by the sequential addition of monomers to ingle subunit counting, the progressive stepwise photo- Significance Sbleaching of fluorescently labeled monomers in a biological complex, has been the method of choice to determine stoichi- Voltage-gated potassium (Kv) channels play a key role in cel- ometries in biological assemblies (1). This technique is based on lular electrical excitability. While various Kv subunits assemble the irreversible photobleaching of green fluorescent protein to homotetrameric functional channels, the silent subfamilies (GFP) tags, which are fused to the protein of interest (Fig. 1 A KvS exclusively form heterotetramers with Kv2 subunits and and F) (1). During prolonged excitation, each GFP progressively thus regulate their biophysical properties in a tissue-specific loses its ability to fluoresce by photochemical destruction, lead- way. Despite the vast functional research, key aspects of the ing to stepwise bleaching events. Since only a fraction of the heterotetrameric architecture remain controversial, including GFPs mature to become fluorescent, one obtains a binomial the stoichiometry with which KvS and Kv2 can assemble. We distribution of N-th order, where N is the number of monomers used concatemers and single subunit counting in combination in the biological assembly. More rigorous evaluation revealed – to show that Kv2/Kv6 assemble in a 2:2 stoichiometry follow- GFP-maturation rates in the range between 0.4 and 0.9 (1 12). ing the general dimer-of-dimer mechanism for channel forma- With increasing N, it becomes difficult to distinguish between the tion. We demonstrate how to objectively choose the most different orders of binomial distributions in this range. Often, likely model for single subunit counting data, which is appli- one must rely on the highest step count; for instance, observation cable for any multimeric complex and will help choosing of a small number of five bleaching steps automatically leads to a models confidently. pentamer and excludes a tetramer. While this logic is theoretically true, it can be misleading if more than one complex colocalize Author contributions: A.J.L., R.B., and D.J.S. designed research; L.M., G.R., and R.B. per- within one diffraction-limited spot. This situation is unavoidable, formed research; L.M., G.R., A.J.L., R.B., and D.J.S. analyzed data; and L.M., G.R., A.J.L., even in fully stochastic distribution of the single complexes. What R.B., and D.J.S. wrote the paper. has been lacking to date is an objective manner of selecting the The authors declare no competing interest. correct model based on a posteriori probabilities. Here, we show This article is a PNAS Direct Submission. how to evaluate different models and use this method to determine Published under the PNAS license. the stoichiometry of Kv2.1/Kv6.4 complexes. 1L.M. and G.R. contributed equally to this work. + Voltage-gated K (Kv) channels regulate the selective flux of 2To whom correspondence may be addressed. Email: [email protected] or dirk. + K ions across the cell membrane by opening, closing, and/or [email protected]. inactivating in response to changes in membrane voltage (13). A This article contains supporting information online at https://www.pnas.org/lookup/suppl/ fully assembled Kv channel consists of four α-subunits, with the doi:10.1073/pnas.1916166117/-/DCSupplemental. NH2 terminus containing the T1 domain as a key determinant in www.pnas.org/cgi/doi/10.1073/pnas.1916166117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 On the other hand, several studies have shown subunit stoi- chiometry of heteromeric Kv channel complexes to vary de- pendent on the relative expression of the different subunits (22–26). It is also important to study only the configuration of functionally expressed Kv2/KvS channel complexes, excluding temporary aggregations that might be formed due to over- expression but that are retained in the endoplasmic reticulum (ER) in a physiological context. Therefore, KvS subunits might be more diverse than assumed, leading to the idea that, in ad- dition to a 3:1 ratio, Kv2/KvS channels assemble in a 2:2 ratio, i.e., as a dimer of dimers. We explored the distribution of stoichiometries with two ex- perimentally independent approaches. To probe the internal arrangement of the different stoichiometries, we compared the biophysical properties of concatemers with corresponding mo- nomeric constructs. With the latter approach, ion-channel stoi- chiometries have been successfully studied (27–29). Possible stoichiometries are 3:1, 2:2, or a population mixing both stoi- chiometries. In single subunit counting experiments, we directly counted the number of Kv6.4 subunits in Kv2.1/Kv6.4 hetero- mers expressed in Xenopus oocytes. Since maturation of GFP, whose photobleaching steps are detected, is not complete, each stoichiometry will lead to a characteristic bleaching-step distri- bution histogram. Choosing the most likely distribution is, therefore, a prominent problem for the analysis and in- terpretation of single subunit counting data. Here, we provide the framework for objectively choosing the most likely (mixture of) stoichiometry of a multimeric arrangement, applicable to any single subunit counting experiments of multimeric complexes. Results Kv2.1/Kv6.4 Heterotetramers Coexpress in a 2:2 Stoichiometry. Kv6.4 is a representative member of the KvS family, given its profound modulating effect on the Kv2 channel properties (30). The direct way to investigate the stoichiometry of Kv2.1/Kv6.4 hetero- tetramers is by single subunit counting experiments, which pro- vide directly the number of fluorescently labeled Kv6.4 subunits Fig. 1. Single-channel subunit counting. (A) Representative frames of a within a single channel assembly. For our studies, we chose the recorded movie of Kv6.4–GFP (Left), Kv2.1–GFP (Center), and Kv2.1/6.4–GFP (Right; 1:8 ratio) expressed in the Xenopus oocyte membrane recorded in Xenopus oocyte expression system for the single subunit counting TIRF configuration. (Scale bars: 2 μm.) (B and D) Representative intensity time experiments because
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