Purification and Structural Study of the Voltage-Sensor Domain of the Human KCNQ1 Potassium Ion Channel

Abstract

KCNQ1 (also known as KV7.1 or KVLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in Escherichia coli and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1–S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 310 component) and underwent rapid backbone amide H–D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure–function experiments.

Influence of Pathogenic Mutations on the Energetics of Translocon-Mediated Bilayer Integration of Transmembrane Helices

“…Based on the crystal structure of bovine rhodopsin (PDB code 1U19), TM7 was judged to correspond to residues 285–309. Based on solution NMR characterization of the voltage sensor domain of KCNQ1 (Peng et al 2014), the S4 helix of KCNQ1 was judged to correspond to residues 221–243. Δ G app,pred and ΔΔ G app,pred values for the wild-type and mutant variants of these helices were calculated using single-helix Δ G predictions with the Δ G prediction server v1.0.…”

Understanding Thermosensitive Transient Receptor Potential Channels as Versatile Polymodal Cellular Sensors

“…Both voltage-sensing and pore domains from other channels have been investigated by solution NMR [79][80][81]. The benefits for solution NMR are that the particularly promising for TRP thermosensing studies.…”

Let’s Twist (the S4) Again

“…Thus, the prokaryotic NavAb sodium channel shows almost the entire S4 in 3 10 conformation, whereas a hybrid a/3 10 conformation is observed in the Kv1.2/2.1 chimera structure (2). Similarly to the VSD of Ci-VSP, the Kv7.1 channel S4 shows a pure a-helix conformation (10). In an attempt to characterize the possible conformational switch of a voltagegated ion channel VSD, the authors push the technique further to test such conformational change for the NavAb channel S4 (11), which is inserted in the structural context of Ci-VSP in two different versions, according to two possible alignments of charged residues.…”

“…When Noda et al (1) cloned the voltage-gated sodium channel, they predicted that a stretch containing charged residues might adopt a 3 10 helix conformation, which was considered rare in proteins. This structure is more tightly wound, longer, and thinner than an a-helix with the same number of residues (2).…”

“…Some excitement came into the field when, as envisioned by Noda et al (1), 3 10 conformations were observed in the available ion channel crystal structures. The characteristics of 3 10 -helices in this structural context may bring some harmony into the S4 controversy, because they would make the S4 tighter-wound, aligning gating charges and favoring their transfer (2) (Fig.…”