Biochemical and structural analysis of the hyperpolarization-activated K(+) channel MVP

Abstract

In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin-spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP.

Figure 1

Canonical Kv and HCN channels share a common VSD and pore domain but are coupled inversely. (A) Schematic showing the shared domains of the voltage-gated ion channel family. (B) Hyperpolarization-activated channels exhibit inverse gating with respect to depolarization-activated channels: they are activated and open at hyperpolarizing potentials, when the conserved voltage sensor is in the down state. (C) GV curves displaying the voltage dependence of a hyperpolarization-activated channel (MVP) and a depolarization-activated channel (Shaker). The curves represent Boltzman fits to experimental data for MVP and Shaker.

Figure 2

Extraction, purification, and characterization of MVP. (A) Western blot showing a detergent screen of MVP-s in pQE60 expressed in XL10 cells. The control is the MVP membrane fraction with no added detergent. MVP appears as two bands differing in oligomeric state (arrows). Although all tested detergents extracted MVP to a certain extent, DDM was chosen for subsequent purification trials, as it appeared to favor the higher oligomeric state. (B) Coomassie brilliant blue-stained SDS–PAGE gel illustrating the two-step purification process of MVP. The last well was overloaded to highlight impurities. (C) Size-exclusion chromatograph of purified MVP in DDM. The elution volume was ∼12 mL on the Superdex 200 HR 10/30 column. (D) MVP’s molecular mass determined by multiangle light scattering methods in DDM.

Figure 3

Reconstitution and stability of MVP in liposomes. (A) FRET analysis of N-terminal, fluorescently labeled MVP indicates that MVP can be reconstituted into many lipids and is most stable in asolectin. The inset shows a cartoon of the FRET assay in which tetramers of MVP are separately labeled with either a fluorescent donor or an acceptor and then combined for reconstitution. The strength of the FRET signal indicates the proximity of MVP tetramers and, by extension, the degree of two-dimensional aggregation. (B) Comparison of changes in FRET levels of MVP in different lipids over time. The change in the FRET level was calculated from the difference in the FRET signal intensity under the experimental condition from that of MVP tetramers in detergent (shown in panel A).

Figure 4

Functional analysis of reconstituted MVP. (A) Traces of MVP-n obtained in symmetric 200 mM KCl (pH 8) at −170 and −200 mV. MVP was reconstituted into asolectin liposomes at a 1:100000 protein: lipid molar ratio. The data were digitized at a sampling rate of 40 kHz and low-pass-filtered to 5 kHz through an eight-pole Bessel filter. Shown below the singles are reconstructions of ensemble measurements from averages of single-channel activity. (B) On kinetics from exponential fits to ensemble currents. (C) Voltage ramp from −200 to 200 mV. Activity at the extreme negative and positive voltages indicates MVP reconstitutes equally in both orientations. (D) I–V curve constructed from single-channel measurements of reconstituted MVP-s in asolectin liposomes.

Figure 5

CW-EPR scan of the S6 helix. (A) Schematic showing the membrane topology of MVP, the region scanned for EPR analysis, and a schematic showing how spin-labels on the S6 helix could lie within the proximity of each other in a tetrameric channel. In the far right panel, the amplitude-normalized spectra for fully labeled (FL) and underlabeled (UL; 1:8 label:cysteine) 195C mutants are shown. (B) CW-EPR spectra of residues 190–196 in POPC/POPG liposomes. This region is located about midway through the S6 helix. Asterisks indicate spectra that exhibit strong spin–spin coupling. (C) Plots of the environmental parameters for the S6 region. Asterisks indicate residues whose spectra showed spin–spin coupling; the arrow denotes the point at which the periodicity in the mobility data ends. Environmental parameters were not obtained for residue 195 because the strong spin–spin coupling interfered with this analysis.

Figure 6

Comparison of KcsA and MVP closed conformations. (A) Alignment of the MVP pore loop and S6 helix (residues 162–209) with the KcsA pore loop and TM2 (residues 67–120). The alignment was made by submitting only the pore TM2 and S6 regions to ClustalW. Asterisks denote residues with strong spin–spin coupling in each channel. The arrow indicates the point at which MVP appears to lose periodicity in its environmental parameters. (B) ΔH₀^–1 for KcsA in the open (pH 3.5) and closed (pH 7) conformations, and MVP. Arrows denote positions with strong spin–spin coupling. (C) Schematic illustrating the differences observed between the closed conformation of KcsA and MVP. Regions of strong spin–spin coupling for each channel are marked with X’s. The channels differ in the region where the strong spin–spin pattern appears, the number of positions with strong spin–spin coupling, and the mobility of the C-terminal ends of the TM2 or S6 segments. This may indicate that MVP’s helical bundle crossing occurs closer to the middle of the membrane.