External Ba²⁺ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter^*

Molecular Biology

cDNA encoding the 411-amino acid isoform of human TREK-1 used in this work was amplified from a TREK-1 plasmid (a generous gift from Dr. Florian Lesage, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France) using PCR. To investigate whether the isoform was under the regulation of alternative translation initiation (ATI) mechanism, TREK-1 containing the translation initial sequence (UGA AUA AGA) preceding the first start codon and the N-terminal truncated isoform (ΔN41) were inserted into pcDNA3.1 vector (Invitrogen). To get a high expression level in the Xenopus laevis oocyte, TREK-1 was subcloned into the pGH19 vector as follows: the pGH19-HERG (human ether-àgo-go-related gene) plasmid (a generous gift of Dr. Gail Robertson, University of Wisconsin-Madison Medical School, Madison, WI) was first cut with BamHI and HindIII to get rid of its initial inserted HERG, and then TREK-1 was inserted into the matched sites.

Mutants were generated using the MutanBEST kit (TaKaRa) according to the manufacturer's manual. All mutations were confirmed by DNA sequencing. cRNA was transcribed in vitro using the RiboMAX^tm large-scale RNA production systems kit (Promega).

Cell Culture, Protein Expression, and Western Blot Analysis

HEK 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 2 mm l-glutamine and held at 37 °C in humidified air with 5% CO₂. cDNAs encoding the wild-type TREK-1, the mutant M42I, and ΔN41 were transiently transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen). Two days after transfection, the cells were harvested. Protein extracts were prepared by solubilization in buffer X (50 mm Tris (pH 7.4), 270 mm NaCl, 1% Triton X) for 1 h and clarified by centrifugation at 12,000 × g for 30 min. Then the lysates were subjected to SDS-PAGE on 10% gel and wet-transferred onto a PVDF membrane. The anti-myc (ZSGB-BIO, 1:1000) and anti-actin (ZSGB-BIO, 1:5000) primary antibodies were used in Western blotting. Secondary antibodies were used at 1:5000 dilutions.

Electrophysiology

X. laevis oocytes were isolated and injected with 10 ng of cRNA (46 nl in volume) per cell. Whole-cell currents were measured 1–3 days after injection by the two-electrode voltage clamp technique using an Axoclamp2B amplifier (Axon Instruments, Union City, CA). For two-electrode voltage clamp experiments, the electrodes were filled with 3 m KCl and had a tip resistance of 0.1–1 MΩ. Recordings were performed under constant perfusion at room temperature. Data were sampled at 2 kHz and filtered at 0.5 kHz with the Clampex 10.0 software (Axon Instruments). K⁺ currents through the TREK-1 channel were elicited either by the Ramp protocol (voltage ramps from −120 to +60 mV, with 1 s in duration) or by the Pulse protocol (a 100-ms pulse from −80 to +20 mV from a holding potential of −80 mV, with a 2 s interpulse interval). The normalized current was the average recorded current divided by the original control current. The standard physiological extracellular solution contained the following unless noted otherwise: 5 mm KCl, 93 mm NaCl, 1 mm MgCl₂, 1.8 mm CaCl₂, 5 mm HEPES (pH 7.4), adjusted with NaOH (standard solution). HEPES buffer was replaced by Tris in pH 8.5 solutions and MES in pH 6.5 solutions. When required, potassium ions in bath solutions were isotonically replaced by sodium ions. BaCl₂ was diluted from a 1 m stock and added to the various solutions as indicated.

Data Analysis

Concentration-response curves were fitted to the Hill equation according to the following parameters: I = 1/(1+ ([X]_o/IC₅₀)^h), where I is the measured current, IC₅₀ is the concentration of extracellular ions ([X]_o, X represents H⁺ or Ba²⁺ in the research) required to achieve 50% inhibition, and h is the Hill coefficient. Time constants were calculated by fitting the data to the following equation: I = I_o+A*e-T/τ, where I is the measured current, I_o is the blocked current at equilibrium, T is the elapsed time after Ba²⁺ application, τ is the apparent forward time constant in seconds, and A is a constant. The fitting was separately performed for each experiment using Origin 7.5 software (OriginLab). Values were presented as mean ± S.E.

Statistical significance was assessed with Student's t test using GraphPad Prism 5.0 (GraphPad Software, Inc.) software. Multiple comparisons were performed with one-way analysis of variance followed by Dunnett post-testing. A significant difference was considered for p < 0.05.

Ba²⁺ Blocks TREK-1 in a Concentration- and Time-dependent Manner

It has been reported that the 426 amino acid of TREK-1 (TREK-1₄₂₆) is subjected to the ATI mechanism because of the suboptimal sequence context flanking the first start codon compared with the second one (34). Therefore, a mixture composed of the full-length TREK-1₄₂₆ and an N-terminal truncated isoform (termed ΔN41 in this study) will be produced by its mRNA (Fig. 1A). The 411-amino acid of TREK-1 (TREK-1), which may be originated from alternative splicing, displayed a very different sequence context surrounding the start site compared with TREK-1₄₂₆ (Fig. 1B). To investigate whether TREK-1 was under the control of the ATI mechanism, a mutant containing the second methionine-to-isoleucine mutation (M42I, no ATI function) was constructed. Protein expression analysis revealed that only one specific band was yielded by wild-type TREK-1 and M42I mutant, whereas the product of ΔN41 was a little smaller than anticipated (Fig. 1C). These results indicate that there is very little, if any, ΔN41 variant that was produced by TREK-1. In other words, the currents produced by TREK-1 would exclusively represent functional expression of TREK-1 rather than a mixture containing ΔN41. Thus, TREK-1 was used in the followed experiments to explore the gate mechanism of SF.

Currents recorded from a representative Xenopus oocyte expressing TREK-1 were shown in Fig. 2A in the absence and presence of different concentrations of extracellular Ba²⁺ ([Ba²⁺]_o). When [Ba²⁺]_o was up to 4 mm, a drastic current reduction was observed compared with the control current. For currents recorded at +20 mV, the inhibition was concentration-dependent (Fig. 2B). The fitting to the Hill equation revealed a half block (IC₅₀) at 0.56 ± 0.03 mm with an apparent Hill coefficient of 1.48 ± 0.18 (n = 7).

Then the time-dependence of the Ba²⁺ inhibition was investigated. In the presence of 1 mm Ba²⁺, the currents evoked by depolarizing from the holding potential of −80 to +20 mV were decreased gradually as the time elapsed until the steady-state blockade was obtained (Fig. 2C). The process was plotted in Fig. 2D, and the current recovery by immediately following Ba²⁺-free solution exchange was also included. The time courses for the block and complete recovery were well fitted by a single exponential equation, with a τ_block of 20.28 ± 1.95 s and a τ_unblock of 45.68 ± 4.80 s (n = 6), respectively.

External K⁺ Impedes the Blocking Effect of Ba²⁺ on TREK-1 Currents

Ba²⁺ inhibition of some potassium channels resulted from its docking properties in the SF (21–23). If that is also the case in TREK-1, competitive binding between Ba²⁺ and K⁺ would occur. To demonstrate the situation, the access rate of Ba²⁺ to the channel was firstly assessed. It was found that the inhibition rate (τ_block, s) was accelerated as [Ba²⁺]_o increased. The τ value declined from 54.32 ± 3.00 s at the 0.25 mm level to 13.18 ± 1.12 s at the 4 mm level (Fig. 3, A and B, n = 6). Then the effects of extracellular K⁺ on Ba²⁺ access were evaluated. When [K⁺]_o was increased from 0 mm to 10 mm, the block time constant at 1 mm [Ba²⁺]_o was prolonged from 14.63 ± 3.88 s to 68.69 ± 10.34 s (p < 0.001, n = 6). Very little access of Ba²⁺ was observed in the presence of 20 mm [K⁺]_o (Fig. 3, C and D). Accordingly, the blocking effects of Ba²⁺ were also alleviated as [K⁺]_o increased. The inhibition was decreased from 93.91 ± 7.41% at the 0 mm [K⁺]_o level to 3.80 ± 0.97% at 20 mm [K⁺]_o level (Fig. 3E, n = 6, p < 0.001). These results reveal the competitive binding between Ba²⁺ and K⁺, which may result from closing binding sites within the SF of TREK-1.

Ba²⁺ Blocks TREK-1 by Overlapped Binding at the S4 Site within the Selectivity Filter

Structural insight into the Streptomyces lividans K⁺ channel, KcsA (potassium channels from S. lividans), provided by crystallographic studies has demonstrated that the binding sites of Ba²⁺ is located in the innermost SF, very close to the K⁺ S4 (9, 35). S4 is composed of four main chain carbonyl oxygen atoms and four threonine side chain hydroxyl oxygen atoms in most potassium channels. The threonine (positions 142 and 251) were conserved in the TREK-1 Pore I and Pore II domains (Fig. 4A). We presumed that Ba²⁺ might occupy a similar position in TREK-1. To test our hypothesis, mutants containing the threonine-to-serine transition at positions 142 (T142S) and 251 (T251S) were constructed. In the presence of 1 mm [Ba²⁺]_o, the T142S and T251S mutants displayed very little blockade (5.97 ± 0.64% and 11.93 ± 2.46% inhibition, respectively, n = 6), whereas the current of the wild-type TREK-1 was decreased dramatically (72.90 ± 4.63% inhibition, n = 7) (Fig. 4B). As the concentration-response curve shows in Fig. 4C, the IC₅₀ of externally applied Ba²⁺ to wild-type TREK-1 was 0.56 ± 0.03 mm, whereas to T142S and T251S it was 3.60 ± 0.19 mm and 3.28 ± 1.20 mm, respectively (n = 6). Obviously, the threonine mutants increase the IC₅₀ for Ba²⁺ by approximately 6 fold, suggesting that Ba²⁺ depresses the TREK-1 current by occupying the K⁺ binding site at the SF.

The Ba²⁺ Exit Rate Uncovers Conformational Alterations within the Selectivity Filter Induced by Different Levels of [K⁺]_o

The SF of KcsA has two distinct conformations associated with low and high concentrations of K⁺. In low-K⁺ solutions, the filter adopts a nonconductive conformation, which is pinched closed at S2 and S3, and K⁺ binds at the ends of the filter (S1 and S4) (36). In high K⁺ solutions, the existence of four fully dehydrated K⁺ ion sites endows the filter in a conductive conformation. According to the well established rule, the alteration of coordinated K⁺ sites would influence the exit of barium from its binding site (35). Therefore, the change of the Ba²⁺ exit pattern would correspond to the conformational alteration of the SF. In our experiments, it was found that the macroscopic currents of TREK-1 were enhanced by increasing [K⁺]_o from 0 to 20 mm (Fig. 5A), implying a conformational transition of the SF from nonconductive to conductive. Then, after the currents were depressed and reached a stable blockade in the presence of 1 mm Ba²⁺, different levels of [K⁺]_o were used to facilitate the exit of Ba²⁺ from its binding site. As shown in Fig. 5, B and C, the exit rate of Ba²⁺ was strongly speeded up as [K⁺]_o increased. The decreased τ values were 45.68 ± 4.80 s, 24.13 ± 1.33 s, and 19.09 ± 5.20 s at 5 mm, 10 mm, and 20 mm [K⁺]_o, respectively (n = 6, p < 0.001). Particularly, the incomplete exit of Ba²⁺ was observed in the K⁺-free external solution (Fig. 5B). Therefore, alteration of [K⁺]_o evokes conformational transitions in the SF of TREK-1, and the off rate of Ba²⁺ is highly associated with the transitions.

External H⁺ Traps Ba²⁺ Inside the Selectivity Filter of TREK-1

TREK-1 is very sensitive to external protons, and acidification strongly inhibits the channel via a mechanism resembling C-type inactivation in voltage-dependent potassium channels (20, 37). The key property of this mechanism is conformational transition of the SF to the nonconductive state. Because structural transitions of the SF occurred between low-K⁺ and high-K⁺ extracellular solution in TREK-1 (Fig. 5), we speculated that the alteration of extracellular pH values (pH_o) might also influence the behavior of Ba²⁺ in response to conformational modifications. In our experiment, the currents of TREK-1 fell quickly after the extracellular solution was switched from pH 8.5 to pH 6.0. Subsequent application of 1 mm Ba²⁺ at pH 6.0 caused a further decrease. However, the washout with pH 6.0 solution achieved only a limited recover of the current (Fig. 6A). High pH_o accelerated Ba²⁺ dissociation with decreased τ_unblock values from 45.68 ± 4.80 s at pH 7.4 to 22.59 ± 3.80 s at pH 8.5 (Fig. 6, B and C, n = 6, p < 0.001). H126A, a pH-insensitive mutant containing a histidine-to-alanine transition (20, 37), was used to eliminate the pH sensitivity of TREK-1. External application of 1 mm Ba²⁺ still blocked the currents quickly, but the currents of H126A recovered completely from Ba²⁺ blockade in pH 6.0 external solutions (Fig. 6D). These data suggest that the conformational transition in TREK-1 occurs in the course of the pH_o switch and that the SF structure evoked by acid tends to trap Ba²⁺ because of its collapsed state.

The Conformational Transitions Induced by Extracellular H⁺ and K⁺-free Are Different in TREK-1

Most recently, it has been demonstrated that the SF conformation caused by low pH is very similar to that caused by low K⁺ in KcsA, of which S2 and S3 are lost (5). In TREK-1, incomplete recovery from Ba²⁺ inhibition was observed in the two conformations evoked by low-pH (acid conformation) and K⁺-free (K⁺-free conformation) conditions. We asked whether the SF structures of TREK-1 caused by K⁺-free and acid conditions are also similar. Our results demonstrate that they are different from each other. Firstly, although the K⁺-free conformation promoted the access of Ba²⁺ (Fig. 3, C and D), the acid conformation tended to impede the process (Fig. 7, A and B). The τ_block was prolonged from 19.14 ± 4.52 s at pH 8.5 to 25.48 ± 1.69 s at pH 6.0 (n = 6, p < 0.01). Next, the interaction between the two conformations was investigated. As shown in Fig. 7C, currents recorded from a representative Xenopus oocyte expressing TREK-1 were subjected to a pH drop from 8.5 to 6.0 in the absence and in the presence of K⁺. Unequivocally, the K⁺-free conformation was more sensitive to the pH drop (I_6.0/I_8.5 of 0.22 ± 0.11 at 20 mV) than the conformation in standard solution (I_6.0/I_8.5 of 0.45 ± 0.12 at 20 mV) (Fig. 7E, n = 5, p < 0.01). Furthermore, the pH-insensitive mutant H126A also showed inhibition by acid in K⁺-free solution (I_6.0/I_8.5 is 0.44 ± 0.10 at 20 mV), compared with that in standard solution (I_6.0/I_8.5 is 0.90 ± 0.10 at 20 mV) (Fig. 7, D and E, n = 6, p < 0.001). Therefore, it seems that the K⁺-free conformation potentiates the formation of acid conformation.

ΔN41 Possesses a Constitutively Collapsed Selectivity Filter

ΔN41, the product of ATI, is permeable to sodium under physiological conditions leading to membrane depolarization (34). The decreased macroscopic current, reduced open probability, along with the alteration of permeability of the isoform reminded us that conformational alteration to nonconductive state at its SF may happen. According to our results, Ba²⁺ ions were trapped in the nonconductive SF induced by low pH_o and K⁺-free conditions in TREK-1. We then employed the blocker to test the possibility. No significant difference was found in the blockade ratio and access rate of Ba²⁺ to wild-type TREK-1 and ΔN41 (Fig. 8, A and C), but the behavior of Ba²⁺ exit displayed a big divergence. Ba²⁺ could not dissociate completely from ΔN41 in standard solution (pH 7.4). In alkali solution (pH 8.5), the time constant (τ_unblock) of ΔN41 was 73.9 ± 10.74 s (n = 7), much bigger than that of the wild type (22.59 ± 3.80 s, n = 6, p < 0.001, Fig. 8, B and C).

To further learn about the conformations of ΔN41, the pH sensitivity of wild-type TREK-1 and ΔN41 was examined next. As shown in Fig. 8D, for the steady-state currents recorded at +20 mV, the IC₅₀ of acid inhibition was 7.29 ± 0.07 and 7.02 ± 0.51 (pH) for wild-type TREK-1 and ΔN41 (n = 5), respectively. No significant difference was found between them (p > 0.05), indicating that the location of conformational alteration induced by acid was similar in wild-type TREK-1 and ΔN41. Meanwhile, the pinched location in ΔN41 was not accordance with that in the wild type induced by acid.