Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy

Abstract

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1–S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

“…Nitroxide-based SDSL EPR power saturation experiments can be used to study the topology of the protein with respect to the membrane [13,57,64,65,70]. There are several biologically important protein systems such as Escherichia coli ferric citrate transporter FecA, vimentin, GM2 activator protein, ABC cassette transporter MsbA, cytochrome C oxidase subunit IV (COX IV), the prokaryotic potassium channel KcsA, KCNQ1-VSD, Pinholin, KCNE1, lactose permease protein, integrin β 1a , functional amyloid Obr2A, C99 domain of the amyloid precursor protein, bacteriorhodopsin, KvAP voltage-sensing domain and phospholamban (PLB), and the GTPase domain of HydF that have been studied using nitroxide-based SDSL CW-EPR spectroscopy to probe the structural, topology, and dynamic properties [51,60,62,65,66,[70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85].…”

“…A recent example of using site-directed spin labeling CW-EPR spectroscopy is the study of human KCNQ1-VSD in proteoliposomes [84]. The human KCNQ1 (Q1) is a voltage-gated potassium channel expressed in several tissues of the body and is known to regulate various physiological functions.…”

“…This study further put together a structural topology model of KCNQ1-VSD in lipid bilayers. Figure 5 shows the proposed topology and the power saturation data on KCNQ1-VSD in lipid bilayers [84]. The CW-EPR power saturation data were analyzed to obtain peak-to-peak amplitude of the first derivative mI = 0 resonance line and to plot against the square root of the incident microwave power for three sample conditions:…”

“…The inset spectra are the corresponding CW-EPR spectra for these sites. (D) Membrane depth parameter (φ) as a function of Q1VSD residue position in POPC/POPG lipid-bilayered vesicles at 295 K. (Adapted from [84] with permission). SDSL CW-EPR spectroscopy at the X-band can also be used to study membrane topology of membrane proteins/peptides bound to aligned phospholipid bilayers [86][87][88][89][90].…”

Palmitoylation of Voltage-Gated Ion Channels

“…Alterations in Kv7.1 activity are also related to sudden infant death syndrome, congenital deafness and familial atrial fibrillation. Because the lipid bilayer is crucial in maintaining the relative orientation of membrane proteins, understanding the structural properties of Kv7.1 in a lipidic environment may be key for the management of pathological consequences [ 115 ]. Mutations in K + channels and their related accessory subunits also participate in different human epileptic phenotypes.…”