| ObjectivePotassium channels form the largest and most diverse function of ion channels, and famous for the characteristic of selectively choosing potassium to pass through. These channels are not only widely expressed in central nervous system, but also in the peripheral system such as skeletal muscle, heart, vessel. They have been implicated in diverse cellular functions, including maintenance of the resting potential and regulation of cellular excitability and muscular contraction. These potassium channels can be divided into four distinct families on the basis of their structure, voltage-gated potassium channel (Kv), calcium-activated potassium channel (Kca2+), inward rectifier potassium channel (Kir) and two-pore domain potassium channel (K2P). Kv, Kca2+and Kir belong to canonical potassium channels which form as tetramer. However, K2P channels are structurally unique in that each subunit possesses four transmembrane segments (TMS1-TMS4) and two pore-forming domains (P1, P2) and form as dimers that allow them to be clearly distinguished from the canonical potassium channels.TREK-2 (TWIK-Related K+ channel) is a member of K2P channel family, which not only expressed highly in central nervous system, but also in the peripheral system. TREK-2 channel conducts leak or background currents that are essential to diverse cellular functions, including maintenance of the resting potential and regulation of excitability, sensory transduction, ion transport, and cell volume regulation, as well as metabolic regulation and apoptosis. The activity of the channel can be regulated by physical factors and natural ligands such as neurotransmitters, GPCR systems, temperature and membrane stretch, as well as by a variety of chemical agents, including intracellular and extracellular protons, polyunsaturated fatty acids, phospholipids and volatile anaesthetics. Above all, TREK-2 plays a key role on the physiological and pathophysiological function through tuning the cellular excitability.Along the ion conduction pathway in canonical potassium channels, there arranged the inner gate (activation gate) and outer gate (also called C-type gate, selectivity filter gate, inactivation gate) from the intracellular side to extracellular side. The mechanism endows potassium channels to effectively control K+ flux, which was referred as gating mechanism. It manipulates permeation pathway of K+ channels by moving the gating elements. There exits three gating mechanisms in potassium channels. The first is outer gating (also called C- type inactivation). Below two conditions will stimulate outer gating. One is that selectivity filter conformation translated from conductive state to nonconductive state, resulting from low concentration of K+, of which the outer gate is collapsed. The other one is that protonation of the pH sensors promotes the collapse of the outer gate. Both of these two conditions are similar to the C-type inactivation of voltage-gated potassium channels. Both of the above mechanisms all induced conformation collape in the outer gate, therefore they are classified as the same mechanism, called outer gating. The second one is inner gating mechanism, which was composed with helices bundle.The glycine hinges which located in the helices bundle are the key motifs to control the K+ flux through conducting the movement of the helice bundle. The third mechanism is termed as N-type inactivation, which is very common in most KV channels. An auto-inhibition of peptide (part of the N terminus of the channel protein) or the assistant subunit, will lead to fast inactivating currents.It has been shown that the inner gate is associated with the outer gate. Outer gate functions through conformational transition, while inner gate’s activity will result in the conformational transition of ion selector, which means there are relationship between the activity of inner gate and outer gate. It has been suggested that the Shaker-KCNKO chimeric channel has the inner gate and outer gate that are different from that of Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in the chimeric channel. However, the chimeric channel is not a native and actual channel. Therefore, it is still unknown whether the two gates are coupled or not.In addition, a lot of evidence indicates that the gating mechanism of potassium is not only regulated by diverse physical and chemical factors, but also by its level of alternative splicing and alternative translation initiation (ATI).ATI is a known mechanism to create protein diversity in prokaryotes and eukaryotic cells. Emerging evidence suggests that proteins generated by ATI can alter subcellular localization and function of transcription and growth factors, hormone receptors, protein kinases and ion channels. In TREK-2, there are three potential initiation sites and the first initiation site is in a suboptimal context for translation initiation while the second is in optimal context, according to the formulation of Kozak. Thus, this may cause ribosomal skipping to occur. Therefore, TREK-2 gene produces long, intermediate and short N-terminus (Nt) isoforms due to ATI mechanism. The Nt isoforms of TREK-2 have similar selectively permeability to K+, but different unitary conductances. However, the role of the Nt in the control of selectivity filter conformation and the relation between unitary conductance and selectivity filter conformation are unknown.In contrast to the rapidly increasing insight into the gating mechanism of potassium channel, the structural basis and molecular mechanisms of TREK-2 channel gating and regulation are still very poorly understood. Some scientific questions still need to be clarified. Here, by using Ba2+, extracellular pH (pHo) and 2-Aminoethoxydiphenyl borate (2-APB) as outer gate and inner gate probing tools, we explore whether the Nt affects the selectivity filter status, and the possible relationship between unitary conductance and selectivity filter conformation. We also explore whether and how the flexibility of glycine hinge and the movements of post-hinge inner helices underlie the gating mechanism of inner gate and outer gate in TREK-2 channels; and what the coupled relationship between the outer gate and inner gate; what the role of C-terminus (Ct) in the gating process.MethodscDNA encoding the human TREK-2 and TREK-1 were cloned into pGH19 vector which could obtain high level expression in Xenopus laevis oocyte. Mutants were generated by using PCR according to different study goals. The concatenated dimeric construct was built by inserting the tandom-linked subunits (wild-type and mutated) into pGH19 vector. The two consecutive subunits was connected by a flexible linker, encoding AAAGSGGSGGSTGGSSGSSGS. All mutations were confirmed by DNA sequencing. Plasmids were linearized by Xho I restriction enzyme then transcribed into cRNA in vitro. Xenopus laevis oocytes were injected with cRNA. Whole-cell currents were measured 1-3 days after injection by Two electrode voltage-clamp (TEVC) technique. K+ currents through TREK-2 channel and all the mutants were elicited by continuous voltage-ramps from-120 to +60 mV from holding potential of-80 mV, with 150 ms in duration (Ramp protocol).2-APB, Ba2+ or different pH extracellular solutions were applied to the channel in various solutions as indicated.ResultsI The inner gate control the outer gate in TREK-2 channeli. The activating mechanisms of 2-APB to TREK-2 channels1.2-APB actives through affecting the C-terminus of TREK-2 channelsIt has been shown that 2-APB activates TREK-2 in a much higher degree compared with that of TREK-1. We suspected that the domains displaying big difference between the two channels might be involved in the 2-APB response on TREK-2. A sequence alignment revealed that the intracellular N-terminus (Nt) and C-terminus (Ct) are the two main lower domains. The Nt deletant (ANt) did not exhibit significant alteration in 2-APB sensitivity compared with that of wild type channels, thus exclude the possibility that the Nt fragment participates in the modulation of 2-APB effects. We then swapped the Cts of TREK-1 and TREK-2, Upon the external application of 2-APB, the Ct of TREK-1 strongly interfered the stimulatory response to TREK-2, whereas the Ct of TREK-2 rescued the stimulatory sensitivity of TREK-1 to 2-APB. These results clearly indicate that the Ct plays a key role in 2-APB response on TREK-2 channels.2. The proximal C-terminus region is required for the interaction between 2-APB and TREK-2, while the distal C-terminus region negatively regulates the interaction.Sequence alignment of Ct between TREK-1 and TREK-2 channels indicated that their main difference locates in the distal Ct (dCt), whereas the proximal Ct (pCt) is relatively conserved. However, the dCt truncated of TREK-2 channel displayed significant enhanced sensitivity upon the application of 2-APB. These results indicated that the pCt region is required for 2-APB binding and the dCt region negatively regulates the interaction between 2-APB and TREK-2.3. His368 located in the pCt region is necessary for 2-APB binding.To evaluate the key residues of this dCt region in 2-APB stimulation on TREK-2, a total of 7 different residues in the pCt region of TREK-2 were substituted to the corresponding ones from TREK-1, and then the sensitivity to 2-APB were evaluated in these mutants. Among them, only H368Y displayed decreased sensitivity to 2-APB which demonstrated His368 plays a dominant role in the opening of the TREK-2 channel induced by 2-APB. His368 may be involved in either the process of binding of 2-APB or gating of TREK-2 channels. In order to find out which process it was involved in, we evaluated the response of H368Y to the extracellular pH (pHo) which mediates the currents of TREK-2 by acting on the SF gate. We found that H368Y displayed no significant alteration in pHo effects compared with WT channels. These results indicated that His368 is essential for 2-APB function but not for pHo effects, implying the residue is required for 2-APB binding, rather than gating of TREK-2 channels.4.2-APB activates TREK-2 channels by manipulating the inner gate.The conformational transition of outer gate induced by changing the concentration of extracellular K+ did not affect the activation of 2-APB to TREK-2 channel. In addition, the putative K+ binding site 4 mutants T172S and T281S which located at the innermost of the selectivity filter exhibited similar behavior upon the application of 2-APB compared with that of WT channels. These data reveal that the status of selectivity filter does not affect the opening of TREK-2 by 2-APB. G312 located in the fourth transmembrane (M4) which composed of the inner gate. And the mutants G312A exhibited significantly decreased sensitivity to 2-APB. These results indicate that at least the M4-Gly hinge is involved in 2-APB- evoked activation, implicating that the compound stimulates TREK-2 activity through manipulating the inner gate.ii. The characteristic of the gating mechanism of TREK-2 channel1. The inner helices act in a subunit-cooperative manner in the gating process of inner gate.Whether the detail mechanism of the interactive pattern of the two subunits in TREK-2 channel during the opening of the inner gate induced by 2-APB is in a subunit-dependent manner or in a subunit-cooperative manner is unknown. The Gly hinge mutations were introduced into the IHs of the concatenated dimmers, including all possible mutated combinations. Note that the double-IH mutation in M2 (G196A monomer) displayed no functional expression, thus all such combination-contained mutants were excluded. We found that the concatenated dimers that harboring Gly hinge mutations within one subunit (a total of six mutants) did not change 2-APB sensitivity significantly. However, the concatenated dimers that harboring Gly hinge mutations in both subunits (a total of five mutants) exhibited decreased sensitivity to 2-APB. These results indicated that in the process of inner gate opening, the two subunits act in a cooperative manner.2. The movements of Gly hinge mediate the conformational transition within the selectivity filter.To identify the function of Gly hinge in the gating mechanism of TREK-2 channels, we examined the response of the Gly hinge-mutants to extracellular pH (pHo) fluctuation. The result is very similar to that of 2-APB response. No significant difference was observed between the Gly hinge mutants (either single-or double-IH) within one subunit. However, the Gly hinge mutants in both subunits (excluding the G196A-G312A and G312A-G196A) exhibited decreased pHo sensitivity. These results indicate that during the conformational transition induced by pHo within the selectivity filter, the movement of the inner gate medicated by the Gly hinge control the outer gate in a subunit-cooperative manner.3. The pCt region controls both gating kinetics of SF gate and inner gate.As established before, the pCt region plays a key role in the function of 2-APB. Because this region is associated with many input modulators in other potassium channels, the function of this region was also examined in the other gating models. Firstly, we found that the deletant that lack of the pCt fragment displayed significantly decreased 2-APB sensitivity. In addition, its response to pHo obviously decreased as well. These results strongly demonstrate that the pCt region is associated with the modulation of the conformational transition of selectivity filter, implying that the domain is also a part of the inner gate and selectivity filter gating apparatus in TREK-2 channels.4. The C-terminus gates both outer gate and inner gate through subunit-cooperativeand cis-type mechanism The pCt region plays a crucial role in the gating mechanism of TREK-2. However, the gating model is still unknown. To address the question, we evaluated 2-APB sensitivity and pHo response of the two tandom-linked dimeric channels composed of a wild type TREK-2 subunit and a truncated molecule short of C-terminus subunit (WT-△Ct and △Ct-WT). We found that both tandems bearing only one intracellular C-termunus exhibited similar 2-APB and pHo response compared with WT tandems. These results indicated that the Ct domain acts in cooperative manner on controlling both gates of TREK-2. In addition, we mutated both Gly hinges of the wild type subunit in △Ct-WT tandems (△Ct-G196AG312A) and found that △Ct-G196AG312A channels showed decreased sensitivity to 2-APB and compared with △Ct-WT. Thus, in the gating processes of both outer gate and inner gate, the normal functional C-terminus and Gly hinge in different subunit is not sufficient to fulfill signal transduction, and Ct controls the motions of inner helices via a cis-type mechanism.5. The movement of inner gate controls the kinetics of outer gateIn conclusion, we found that the mutants involved in the Gly hinge and C-terminus showed decreased sensitivity not only to 2-APB, but also to pHo. Both the Gly hinges and proximal C-terminus are the key motif in controlling the inner gate, thus, we noted that the movement of inner gate controls the kinetics of outer gate in the gating mechanism of TREK-2 channel.Ⅱ The isoforms generated by alternative translation initiation mechanism adopt similar conformation in the selectivity filter in TREK-2 channelIt has been established that Ba2+ is a useful tool to explore the conformation of the selectivity filter in potassium channels. Since the selectivity filter regions are highly conserved in most potassium channels, we examined the response of theTREK-2 channel to Ba2+.1. We found that Ba2+ inhibits TREK-2 currents in a concentration- and time-dependent manner. And the inhibition of Ba2+ to TREK-2 channels is strongly affected by the concentration of extracellular K+. In addition, the putative K+ binding site 4 mutants T172S and T281S which located at the innermost of the selectivity filter showed dramatically decreased sensitivity to Ba2+. These results indicated that Ba2+ blocks the TREK-2 current by occupying the K+ binding site 4 within the selectivity filter. Thus, Ba2+ is able to be used as specific tools to define the conformational alteration in S4 site.2. T172S and T281S showed similar pHo response with the wild type TREK-2 channels in the physical concentration K+ which indicated that the conformational transition evoked by pHo fluctuation is other than the S4 site, that is, the outer position of selectivity filter.3. No significant difference was found in the inhibition, access and exit rate of Ba2+ among ATI isoforms which demonstrated that these isoforms roughly adopt similar conformation in S4 site of selectivity filter. Meanwhile, the ATI isoforms experienced similar conformational transitions induced by pHo indicated that all ATI isoforms possess similar conformation in the outer of the outer gate. These results revealed that the ATI isoforms possess similar conformation in the outer gate. On the other hand, the N-terminus plays a little role in the conformational transition of the outer gate.ConclusionIn this study, we characterized the activation of 2-APB on TREK-2 channel and the gating kinetics of TREK-2 channels. Firstly, we found that 2-APB binding on the His368 which was located in the proximal Ct stimulates the activity of TREK-2 channels via manipulating the inner gate. We then used the compound and pHo as probing tools to explore the inner gate and outer gate, respectively. And we found that the Gly hinges and Ct especially the proximal region are the key motifs in controlling the both inner and outer gates. The two subunits act in a cooperative manner in the gating process. The interaction manner between C-terminus and the Gly hinges is cis-type mechanism. In the gating process of TREK-2 channel, the inner gate was the in the leading position and its movement controls the kinetics of the outer gate.Ba2+ blocks TREK-2 channel by overlapped binding at the putative K+ binding site 4. And using Ba2+ and pHo as probing tools, we found that the isoforms generated by alternative translation initiation mechanism adopt similar conformation in the selectivity filter in TREK-2 channel.Our study reveals a new gating mechanism of two-pore domain potassium channel, and provides a new theory for the drug design and development. In addition,2-APB, Ba2+ and pHo could be used as probes to explore the function and structure of TREK-2 channel and the other potassium channels. |
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