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. 2011 Nov 18;286(46):39813-22.
doi: 10.1074/jbc.M111.264788. Epub 2011 Sep 29.

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

Xiao-Yun Ma  1 , Jin-Mei Yu, Shu-Zhuo Zhang, Xiao-Yan Liu, Bao-Hong Wu, Xiao-Li Wei, Jia-Qing Yan, Hong-Liang Sun, Hai-Tao Yan, Jian-Quan Zheng
Affiliations

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

Xiao-Yun Ma et al. J Biol Chem. .

Abstract

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of the TREK-1 isoform. A, comparison of protein sequences of ΔN41, TREK-1, and TREK-1426. The second methionine in TREK-1 and TREK-1426 are boxed. B, comparison of mRNA sequences flanking the first (M1) and second (M42) start codon of the TREK-1, the optimal Kozak sequence (Optimal), and the first start codon (ATI) of TREK-1426. The start codons are boxed. C, protein expression analysis of ΔN41, the M42I mutant, and wild-type TREK-1 by Western blot analysis with anti-myc antibody. The internal control (Actin) was visualized by an anti-actin antibody.
FIGURE 2.
FIGURE 2.
TREK-1 channels are inhibited by externally applied Ba2+ in a concentration- and time-dependent manner. A, representative current-voltage relationship for TREK-1 expressed in Xenopus oocytes in the absence and presence of different [Ba2+]o. Currents were elicited by the ramp protocol. The concentration of K+ in extracellular solution is 5 mm. B, concentration-response for Ba2+ inhibition (%) of TREK-1 channels at 20 mV with the protocol in A. Data points were mean ± S.E. of seven cells. The solid line is a fit of the data to the Hill equation. C, Ba2+ block of representative TREK-1 current amplitude during application 1 mm Ba2+. Currents were recorded with the pulse protocol. Io is the original current before application of Ba2+. D, plot of current-time recorded from the currents recorded in C. The following withdrawal of Ba2+ was also included.
FIGURE 3.
FIGURE 3.
Effects of different [K+]o on Ba2+ inhibition of TREK-1 currents. A, the time course of inhibition to TREK-1 channels by different [Ba2+]o from a representative TREK-1-expressing oocyte. B, the time constant (τblock, s) of inhibition to TREK-1 current by different [Ba2+]o (mean ± S.E., n = 6 cells). ***, p < 0.001 versus 0.25 mm [Ba2+]o. C, effects of different [K+]o on the time course of blockade by 1 mm Ba2+ externally applied to a representative oocyte expressing TREK-1 channels. D, the block rate (τblock) (mean ± S.E., n = 6) of Ba2+ in different [K+]o external solutions as indicated. ***, p < 0.001 versus 0 mm [K+]o. The block rate was not significantly different at 0 and 5 mm [K+]o (p > 0.05). E, the inhibition percentage to TREK-1 current by Ba2+ in different [K+]o. Data represent mean ± S.E.(n = 6). ***, p < 0.001 versus 0 mm [K+]o.
FIGURE 4.
FIGURE 4.
Ba2+ inhibits TREK-1 current through binding at the location occupied by Threonine 142 and Threonine 251 in the SF. A, multiple sequence alignment of the SF regions of TREK-1 (P1 and P2), KcsA, Kir2.1, and Kv1.5. The positions of the conserved threonine residues among them are boxed. The SF GY/FG signature sequence is also boxed. B, comparison of the relative 1 mm Ba2+ inhibitory effects on wild-type TREK-1, the T142S mutant, and the T251S mutant. The currents recorded from representative oocytes expressing the above channels were recorded, respectively. C, concentration-response curves of the wild-type TREK-1 and the mutants. Data points were mean ± S.E. for six to seven cells. The data were fitted with the Hill equation.
FIGURE 5.
FIGURE 5.
Effects of different [K+]o on the dissociation of Ba2+ from the TREK-1 channel. A, effects of different [K+]o on the macroscopic currents of TREK-1 recorded from a representative oocyte. B, effects of different [K+]o on the time course of recovery from the blockade by 1 mm Ba2+ in a representative TREK-1-expressing oocyte. C, the time constant (τunblock, s) (mean ± S.E., n = 6) of Ba2+ inhibition in different [K+]o as indicated. ***, p < 0.001 versus 5 mm [K+]o.
FIGURE 6.
FIGURE 6.
Effects of different pHo on the exit of Ba2+ from the TREK-1 channel. A, acidic pHo inhibits Ba2+ unbinding from TREK-1. Experiments were started in pH 8.5 bath solution. The external solution was switched between pH 8.5 and pH 6.0, as indicated. ↓ and ↑ indicate the time of Ba2+ (1 mm) fast application and withdrawal, respectively. B, the time course of Ba2+ exit from TREK-1 in different pHo solutions. The representative TREK-1 currents were depressed by 1 mm Ba2+ in standard solution and then subjected to washout by different pHo solutions. C, time constant (τunblock) of Ba2+ exit from TREK-1 channels in different pHo. Data represent mean percentage ± S.E. (n = 6). ***, p < 0.001 versus pH 7.4. D, Ba2+ dissociates completely from the H126A mutant in pH 6.0 external solutions. Currents were recorded from a representative H126A-expressing oocyte. ↓ and ↑ indicated the time of Ba2+ (1 mm) fast application and withdrawal, respectively.
FIGURE 7.
FIGURE 7.
Characterization of the conformational modifications induced by K+-free and acid conditions in the selectivity filter of TREK-1. A, the time course of Ba2+ access to TREK-1. Currents were recorded from a representative TREK-1-expressing oocyte and were subjected to 1 mm Ba2+ perfusion in different pHo solutions. B, time constant (τblock) of Ba2+ access to TREK-1 channels in different pHo solutions. Data represent mean percentage ± S.E. (n = 6). **, p < 0.01 versus pH 8.5. τblock was not significantly different at pH 8.5 and 7.4 (p > 0.05). C, pHo effects on the representative TREK-1 currents in the standard and K+-free external solutions. D, pHo effects on the representative currents for H126A mutant expressed in Xenopus oocytes in the standard and K+-free external solutions. E, comparative analysis of I6.0/I8.5 in wild-type TREK-1 and the H126A mutant in different [K+]o. Data represent mean fraction ± S.E. (n = 5–6) at 20 mV. **, p < 0.01 versus wild-type at 5 mm [K+]o. ***, p < 0.001 versus H126A at 5 mm [K+]o.
FIGURE 8.
FIGURE 8.
Identification of the selectivity filter status in ΔN41. A, the time course of Ba2+ access to the representative wild-type TREK-1 and ΔN41 channels. B, the time course of Ba2+ dissociation from wild-type TREK-1 and ΔN41 channels. C, comparative analysis of time constants (τ, s) of Ba2+ blocking and unblocking in wild-type TREK-1 and ΔN41 channels. Data are mean ± S.E. from five to seven cells. τblock was not significantly different between the wild type and ΔN41 at pH 7.4 (p > 0.05). ***, p < 0.001 versus the wild type at pH 8.5. D, comparison of response to different pHo between wild-type TREK-1 and ΔN41. Data points represent mean fraction ± S.E. (n = 5) at 20 mV.
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