Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

Abstract

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

KCNQ and KCNE Isoform-Dependent Pharmacology Rationalizes Native American Dual Use of Specific Plants as Both Analgesics and Gastrointestinal Therapeutics

“…Patch-clamp electrophysiology experiments were performed as before ( Manville et al, 2018 ; Harkcom et al, 2019 ; Tedeschi et al, 2021 ) on CHO cells at room temperature (22–25°C). Using a whole-cell patch configuration under voltage-clamp conditions, data were obtained with an Axopatch Multiclamp 700A apparatus digitized and analyzed using pClamp 9.2 (Axon Instruments, Forster City, CA), together with Graphpad Prism 9 (Graphpad; La Jolla, CA, United States).…”

“…Patch-clamp electrophysiology experiments were performed as before Harkcom et al, 2019;Tedeschi et al, 2021) on CHO cells at room temperature (22-25 (Hampton, NH) or Sigma-Aldrich. Tannic acid was added to the extracellular bath solution at concentrations between 1 and 10 µM.…”

Measuring protein-membrane interaction through radial fluorescence correlation in 2 dimensions

“…As previously described by Torrado [23] and Tedeschi [24], NIH/3T3 (ATCC) cells were cultured in Dulbecco's modified Eagle medium (Life) supplemented with 10% v/v fetal bovine serum (FBS, Life), and 1% v/v Penicillin/Streptomycin Solution 100X (10,000 units of penicillin and 10mg/mL streptomycin in 0.85% saline solution, GenClone, Genesee) at 37 °C in a 5% CO2 incubator. For the mEGFP control, we added to the medium, 3 μl/ml of Hygromycin to encourage the expression of mEGFP.…”

“…As an experimental verification of our capacity to detect the interaction strength between protein and membranes, we performed a 2D-pCF analysis on cells transfected with GAP-mEGFP. This construct is the membrane-bound form of mEGFP and as is not functional, its diffusion is only dictated by the surrounding environment [24]. We could expect GAP-mEGFP to have a greater anisotropy gap around the plasmatic membrane compared to that of mEGFP.…”

Comparison of MSD analysis from single particle tracking with MSD from images. Getting the best of both worlds

“…These methods can evaluate modes of motion by analyzing the time dependence of the fluorescence intensity correlation in a stack of images, without the need for localization. These properties make iMSD a powerful technique for studying relevant biological problems, such as the diverse intracellular dynamic processes and the viscosity of the cell interior [6,[10][11][12][13][14][15][16][17]. As with any other fluctuation-based analysis method, iMSD provides average information about all the diffusing elements in the image stack, allowing good statistics in a limited amount of time [10].…”