Letter pubs.acs.org/JPCL
Gating-Associated Clustering−Dispersion Dynamics of the KcsA Potassium Channel in a Lipid Membrane Ayumi Sumino,†,‡ Daisuke Yamamoto,§ Masayuki Iwamoto,‡ Takehisa Dewa,∥ and Shigetoshi Oiki*,‡ †
PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuokashimoaizuki, Yoshida-gun, Fukui 910-1193, Japan § Department of Applied Physics, Fukuoka University, 8-19-1 Nanakuma, Fukuoka 814-0180, Japan ∥ Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan ‡
S Supporting Information *
ABSTRACT: The KcsA potassium channel is a prototypical channel of bacterial origin, and the mechanism underlying the pH-dependent gating has been studied extensively. With the high-resolution atomic force microscopy (AFM), we have resolved functional open and closed gates of the KcsA channel under the membrane-embedded condition. Here we surprisingly found that the pH-dependent gating of the KcsA channels was associated with clustering−dispersion dynamics. At neutral pH, the resting, closed channels were coalesced, forming nanoclusters. At acidic pH, the open-gated channels were dispersed as singly isolated channels. Time-lapse AFM revealed reversible clustering−dispersion transitions upon pH changes. At acidic equilibrium, a small fraction of the channels was nanoclustered, in which the gate was apparently closed. Thus, it is suggested that opening of the gate and the dispersion are tightly linked. The interplay between the intramolecular conformational change and the supramolecular clustering− dispersion dynamics provides insights into understanding of unprecedented functional cooperativity of channels. SECTION: Biophysical Chemistry and Biomolecules
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measurements, and effects of lipid composition and phase transitions have been studied.13−16 Methods for detecting the localization of channel proteins in membranes as well as approaches for detecting the gating conformational changes with sufficiently high spatial resolution are prerequisite to further examine the microscopic process of channel proteins. Recent developments in atomic force microscopy (AFM) allow for topological mapping of membrane proteins in the membrane-embedded condition,17−22 and different conformations of membrane proteins have been detected.23−28 In this study, we focus on the dynamic behavior of the KcsA channel on the lipid membrane. The KcsA channel is a prototypical potassium channel of bacterial origin, and the transmembrane pore domain shares a common architecture with others in the potassium channel gene family.29 The KcsA channel exhibits pH-dependent gating, and the mechanism underlying the gating has been studied extensively.30−33 Structural studies of the KcsA channel have provided insights into the molecular mechanism of channel function, while the structural data were mostly attained from the solubilized
any membrane proteins diffuse more or less freely within a restricted area of a fluidic membrane and encounter and interact with proteins of the same or different molecular species.1,2 With this dynamic behavior, membrane proteins exhibit specific modes of interplay, leading to a variety of signal transduction events.3−6 For the ion channel, the interactions with signal transduction molecules have been studied extensively,7,8 whereas interactions among the same channel species remain unclear. Channels are sometimes localized with high density in a restricted area of the cell membrane, where they are close enough to interact. However, functional measurements revealed that cellular macroscopic currents are generally a linear sum of the single-channel currents.9 In addition to static distributions of channel proteins on membranes, diffusion of channels allows reversible and temporal interactions. Dynamic behavior of channel proteins on the cell membrane still remains elusive. To examine close interplay among channels, researchers have performed experiments under more controllable systems. In the heterogeneous expression system, it has been revealed that the macroscopic characteristics of channel currents, such as the gating kinetics, change when the density of expressed channels in a membrane is altered.10−12 In liposome membranes, the clustering of channels has been observed using fluorescence © 2014 American Chemical Society
Received: November 18, 2013 Accepted: January 20, 2014 Published: January 20, 2014 578
dx.doi.org/10.1021/jz402491t | J. Phys. Chem. Lett. 2014, 5, 578−584
The Journal of Physical Chemistry Letters
Letter
channel proteins. We have demonstrated gating conformational changes of the KcsA channel in a membrane-embedded condition using AFM.28 To gain access to gating structural changes with the AFM probe, we removed the potentially sightobstructing cytoplasmic domain (CPD) (CPD-truncated channel), while the channel retained the pH-dependent gating. AFM revealed the closed conformation at neutral pH as a small protrusion of the sensory domain from the cytoplasmic membrane surface. At acidic pH, the open-gate structure was resolved at the center of the tetrameric channel.28 Upon opening, the transmembrane domain (TMD) reduced its longitudinal length due to the global twisting motion,34 which may render changes in the channel−lipid interaction. Here we present the clustering and dispersion of KcsA channels in the membrane; these processes are associated with pH-dependent gating conformational changes. The gating status (i.e., open or closed) and the collective behavior of clustering and dispersion in the membrane were concurrently resolved at the single-molecule level using high-resolution AFM. Furthermore, time-lapsed AFM images elucidated the dynamics of the channel motions in the membrane that led to clustering and dispersion. The gating-associated clustering− dispersion dynamics represent an unprecedented mode of channel activity relevant to physiological function on cell membranes. Moreover, the present finding sheds light on the physicochemical events occurring at the channel−lipid interface. Formation of KcsA Channel Clusters at Neutral pH. Figure 1 shows AFM images of a CPD-truncated KcsA channel in a membrane under neutral (A,C,E) and acidic conditions (B,D,F). For AFM observation, KcsA-incorporated liposomes were extended on the mica surface, and membrane islands formed; these islands were distinguished as a terrace on the flat mica surface. The channels were found exclusively in the membrane islands as small protrusions from the membrane surface formed by the cytoplasmic end of the TMD. The bright areas found on the membrane surface, indicating a height of more than several tens of nanometers from the mica surface, represent lipid−protein aggregates in the membrane and were ignored hereafter. At neutral pH, the KcsA channels were coalesced, forming small patches or self-assembled nanoclusters in the membrane (Figure 1A). Isolated channels were scarcely observed outside the nanoclusters (Figure 1C). These nanoclusters were small, and the size did not increase further after several hours at neutral pH. Similar clusters were also found when the lipid/ protein ratio was increased to 100/1 (w/w) from the standard ratio of 3 to 5/1 (Supplementary Figure S1 in the Supporting Information). Some channels were distributed at the edge of the membrane islands, forming a narrow boundary fence. The nanocluster formation in the membrane and the fence formation at the edge occurred exclusively at neutral pH (see the following section; Supplementary Figure S2 in the Supporting Information). These collective behaviors should share common physicochemical features in terms of channel− membrane interactions,35 even though the membrane islands formed on the mica for AFM measurements are artificial. A magnified view at neutral pH demonstrates several distinct particles in each nanocluster (Figure 1C); these particles correspond to individual channels with a longitudinal length of 6.4 nm (Figure 1A, lower panel) and a width of 6 to 7 nm. The channels were closely packed, but a 2-D lattice arrangement was not detected in the cluster. The height profile (Figure 1A
Figure 1. Clustering and dispersion status of KcsA channels in the membrane, observed by AFM. AFM images of CPD-truncated KcsA channels in lipid bilayers at pH 7.5 (A,C,E) and pH 4.0 (B,D,F). KcsAreconstituted proteoliposomes were attached and extended onto the mica surface, forming a flat bilayer. The cytoplasmic region of the KcsA channel protruded from the membrane surface. The pH of the overlaying buffer solution was 7.5 for panels A,C,E and 4.0 for panels B,D,F. The height profiles along the white dashed lines are shown below the images. The reference height level (the zero level) is set at either the mica surface (A,B) or the membrane surface (C,D). The images were recorded using acoustic AC-mode AFM. The inset in panel D shows an averaged image of KcsA (N = 23, full scale: 7 nm). 3-D profiles of the channel in the membrane near the membrane edge are shown at pH 7.5 (E) and 4.0 (F).
lower) and the 3-D rendering image (Figure 1E) indicate that the channels in the nanoclusters and those at the membrane boundary share a similar longitudinal length that is characteristic of the closed conformation (see Figure 5B).28 The footprint of the nanoclusters was measured using the AFM images; the histogram (Figure 2A) shows a broad distribution, but extremely large clusters (>1500 nm2) were rarely observed. In the clusters, the average distance between the nearest peaks of the height profile was found to be 10 nm (Figure 2B). A rough estimate of the average number of channels in a cluster 579
dx.doi.org/10.1021/jz402491t | J. Phys. Chem. Lett. 2014, 5, 578−584
The Journal of Physical Chemistry Letters
Letter
channels. These distributions suggest the existence of a repulsive force between channels. pH-Dependent Supra-Molecular Assembly in Liposomes. In a flat membrane attached to the surface of mica, 2-D diffusion of membrane proteins is generally restricted. To examine the formation of nanoclusters on a membrane that allows free diffusion, KcsA channels in liposomes were incubated in either acidic or neutral pH, and SDS-PAGE was performed (Figure 3). As a control experiment, detergent-solubilized channels
Figure 2. Cluster size in the lipid bilayer and nearest-neighbor distance of the KcsA channel. (A) Footprint areas of channels in clusters and in isolated single channels. The area of clusters or individual channels above a defined height was measured using the AFM image (pH 7.5, green, N = 188; pH 4.0 all, orange, N = 1199; pH 4.0 clustered, cyan, N = 185). Inset: The footprint areas of clusters. A fraction of channels forms clusters even at acidic pH, and the area was compared with that of the cluster at neutral pH. (B) Nearest-neighbor distances. The distance was measured from center to center of the KcsA channels (pH 7.5, green, N = 101; pH 4.0 isolated, orange, N = 1007; pH 4.0 clustered, cyan, N = 105). Inset: The nearest-neighbor distance of channels in the cluster.
Figure 3. Supramolecular assembly of the KcsA channel on the liposome membrane. SDS-PAGE profiles of KcsA channels in the detergent-solubilized form and in liposomes at acidic and neutral pH. KcsA channels in n-dodecyl-β-D-maltoside (DDM) and in liposomes were incubated at pH 4.0 and 7.5 for several hours prior to SDSPAGE. M-T and D-T indicate single (52 kDa) and double (104 kDa) tetramers of KcsA, respectively. The D-T band is enhanced at the lane for liposomes at pH 7.5, indicating that the lipid bilayer environment and neutral pH facilitate clustering of the KcsA channel.
based on the average distance is
