科学研究費助成事業　　研究成果報告書

様　式　Ｃ－１９、Ｆ－１９－１、Ｚ－１９ （共通）

機関番号：

研究種目：

課題番号：

研究課題名（和文）

研究代表者

研究課題名（英文）

交付決定額（研究期間全体）：（直接経費）

３２６８９

若手研究(B)

2016～2014

Conformational Control of Ion Channels

Conformational Control of Ion Channels

８０５９６２２１研究者番号：

ＢＥＲＴＺ　Ｍ（Bertz, Morten）

早稲田大学・ナノ・ライフ創新研究機構・次席研究員（研究院講師）

研究期間：

２６８７０６４２

平成 年 月 日現在２９ ６ １５

円 2,800,000

研究成果の概要（和文）：The voltage sensor domain (VSD) of ion channels transmits its conformational change through flexible linkers.Here, we find that the length of N-terminal linker controls the conformational space of the VSD while the C-terminal linker transmits the conformational change to the pore.

研究成果の概要（英文）：The voltage sensor domain (VSD) of ion channels senses changes in transmembrane potential and triggers opening and closing of the channel's pore.The conformational change of the VSD is transduced to the channels pore through flexible linkers which ultimately control the VSD’s resting and activated conformation. In this project, we have investigated the influence of both length and composition of the N- (S3S4) and C-terminal (S4S5) VSD linkers on BK channel gating and activation.We find that only the length but not the composition of the N-terminal linker controls the conformational space available to the VSD, with profound effects on gating kinetics. The C-terminal linker is responsible for transmitting the conformational change to the pore, and even minor mutations significantly diminish voltage sensitivity. Some degree of voltage sensitivity, however, is retained, which is in accordance with recently available structural data.

研究分野： Electrophysiology

キーワード： ion channel　single-molecule

１版

( Voltage gating - the reaction of ion channels to changes in membrane potential - is fundamental to signal transduction in living organisms. In many ion channels, voltage sensor domains containing conserved positively charged residues move according to the transmembrane electrical field. Electromechanical coupling between voltage sensor movement and pore opening is, at least in part, achieved through flexible linkers that connect the mobile voltage sensor both N- and C-terminally to the rest of the channel protein. These linkers transmit the conformational change of the voltage sensor to the pore, control the sensor’s resting position, and ultimately limit the range of travel of the voltage sensor during activation.

( In voltage-gated ion channels, opening and closing of the channel’s ion-conducting pore is triggered by changes in transmembrane potential. Conserved positively charged residues in the S4 helix of the voltage sensor move in reaction to the external electrical field and this voltage-dependent conformational change is transmitted to the channel pore. In a simple model, depolarization (activation/opening) moves S4 outwards towards the extracellular side of the membrane, while hyperpolarization (deactivation/ closing) causes S4 to slide inwards (Fig. 1A).

Figure 1 In this model, the range of travel of S4 is ultimately limited by its N and C terminal linkers to helices S3 (S3S4) and S5 (S4S5). Peculiarly, in BK channel, the S3S4 linker consists of merely three residues, the minimum number required to connect two α-helices, in contrast to canonical voltage-gated ion channels that feature much longer linkers. Here, we systematically vary the length and composition of both S3S4 and S4S5 to

investigate the linker’s influence on BK channel gating and activation (Fig. 1B).

( The influence of the S3S4 linker on murine BK channel gating was investigated using mutants with glycine / serine insertions of 1, 2, 3, 6, 10, and 15 residues in length. As a control, individual linker residues or the entire S3S4 linker were replaced by glycine. Correspondingly, glycine / serine linkers of 1, 2, 3, 6, 10, and 15 amino acids in length were inserted into S4S5 at the C-terminal tip of S4 (Fig. 2). Voltage-dependent channel opening and closing were studied using two voltage protocols, and opening or closing kinetics were determined by single-exponential fits to the channel current time course. In addition, the voltage-dependence of channel activation was determined from tailcurrents at -80 mV. All experiments were done in inside-out patches at 5 µM Ca2+ (buffered with EGTA) for insertions in S3S4 and 500 µM Ca2+ for insertions in S4S5 with symmetrical [K+]=150 mM.

Figure 2

We find that while additional residues in S3S4 are well tolerated, insertion of even a few amino acids markedly slows down channel opening and closing.

Figure 3

The effect of insertions on the midpoint of activation Vhalf, however, is very small, with a maximum shift of less than 10 mV and no clearly discernible trend in the cooperativity of activation (Figure 3 A). The composition of S3S4, however, turned out to be inconsequential, and replacing the entire linker (wild-type sequence NRS) with glycine had no discernible effect on channel kinetics or voltage sensitivity (dark red data points in figure 3 C-E). The length of this linker, however, was of crucial importance, and no voltage-dependent currents could be elicited from mutant channels with single or all linker residues removed by mutation (not shown). Fluorescence tagging revealed that truncation of the S3S4 linker likely impedes channel folding and assembly, or subsequent membrane trafficking (not shown).

Figure 4 Figure 4 summarizes these results. The length of the S3S4 linker has profound effects on the kinetics of channel opening and closing. Opening and closing kinetics slows down gradually starting from even one inserted residue. The voltage dependence of opening and closing kinetics, which depends on the number of charges and the degree of their displacement in each transition, shows two effects: While opening kinetics slowly become more voltage-sensitive (Figure 3E), closing kinetics show a drastically reduced voltage sensitivity for insertions longer than six residues (Figure 3C). Replacing individual or even all S3S4 linker residues with glycine results in channels with properties very similar to the wild-type channel, illustrating that the observed effect on channel kinetics is caused by linker length and not by changes in linker sequence. We find that the length of the S3S4 linker limits the travel and conformational space of the voltage-sensing S4 helix. Glycince-serine insertions at the N-terminal end of S4S5 were well-tolerated by BK. While mutant channels retained voltage sensitivity, the midpoint of activation Vhalf was shifted by ≈ +200 mV to

depolarizing voltages at 5 µM [Ca2+] compared to the wild-type (not shown). Increasing [Ca2+] to 500 µM shifted Vhalf of the mutant channels into the experimentally accessible range. Even short insertions of a single glycince residue into S4S5 had a significant effect on voltage gating: Vhalf was shifted by ≈ +60 mV (at [Ca2+] = 500 µM), the opening rate decreased, and the closing rate increased.

Figure 5 Intriguingly, mutants caused the closing rate losing most of its voltage dependence (Figure 5A&B). These effects all increased gradually with longer glycine-serine insertions up to 6 residues. For even longer insertions (10 and 15 residues), the shift of Vhalf was partially reversed and decreased by ≈-50 mV for the longest linker compared to +6 inserted residues (Figure 5 A&C). This reversal was mostly due to a faster opening rate (Figure 5B top). The voltage-dependence of the opening transition remained unaffected by insertions. Apparently, even short insertions are sufficient to disrupt the secondary / tertiary structure of S4S5, thereby impeding the transmission of the conformational change of S4 to the pore. Signaling through S4S5 might be disrupted entirely, and the pore is opened through a secondary pathway independent of S4S5 (Figure 6A). In this scenario, the channel closes when stress on the pore is relaxed, more or less independent of voltage.

Figure 6 Insertions >6 residues in turn facilitate opening compared to short or medium length insertions. Bulky insertions might facilitate opening the channel via an S4S5-independent pathway involving interactions of the tip of S4 and the intracellular RCK domain of BK (Figure 6B). Such a mechanism has been proposed in recent structural studies (Tao, Nature 2017).

1)

3 1. 52nd Meeting of the Biophysical

Society of Japan, Sapporo, Japan (9/24/2014-9/27/2014)

Morten Bertz & Kazuhiko Kinosita, Jr.: Conformational Transitions in Voltage Sensor Domains

2. 53nd Meeting of the Biophysical Society of Japan, Kanazawa, Japan (9/12/2015-9/15/2015) Morten Bertz & Kazuhiko Kinosita,

Jr.: Conformational Control of Voltage Sensor Domains

3. The 60th Annual Meeting of the Biophysical Society, Los Angeles, USA (27/2/2016-3/3/2016) Morten Bertz & Kazuhiko Kinosita,

Jr.: Conformational Control of Voltage Sensor Domains

BERTZ, Morten Waseda University, Institute for Nanoscience & Nanotechnology (Lecturer)

80596221