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

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 timedependent 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 (⌬N41), 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.Potassium channels are ubiquitous pore-forming transmembrane proteins that transport K ϩ ions selectively and rapidly across the biological membranes. The efflux of K ϩ ions is controlled not only by the electrochemical gradient, but also by the gating mechanism. Along the ion conduction pathway of potassium channels, three structures are arranged from intercellular to extracellular: the lower activation gate, the selectivity filter (SF) 2 , and the upper inactivation gate (also termed the C-type inactivation gate). Correspondingly, there are mainly two kinds of mechanisms controlling K ϩ ion passage. Manipulation of the lower activation gate controls the transition between the open and close state of the channel. The upper inactivation gate, which is characterized by slow kinetics, controls the transition between conduction and nonconduction of the pore. The selectivity filter of the K ϩ channels, formed by the highly conserved sequence TV(I)GY(F)G, plays a pivotal role in both mechanisms. Accumulating evidence shows that the selectivity filter itself has the ability to act as the inactivation gate (1-4). High resolution crystallographic analysis has revealed detailed structural changes in the selectivity filter associated with the activation gating and inactivation gating (5, 6). The carbonyl oxygens together with the side chain hydroxyl oxygen of the threonine define four equally spaced ion-binding sites that are commonly termed S1-S4, from the extracellular to the intracellular region (7)...

Two-pore domain potassium channels: potential therapeutic targets for the treatment of pain

“…The alternative translation initiation codon is 42 amino acids upstream from the first methionine in the sequence at the N-terminus of the channel. The N-terminus truncated form gives rise to a current with a much reduced open probability and a collapsed selectivity filter with both a reduced potassium selectivity [61,74,94,100] and a measurable permeability to sodium [94]. Treatment with both DEPC and fenamates, substantially enhances current through the Nterminus truncated form of the channel, and, interestingly, measurements of the reversal potential in the presence of these compounds reveal that the enhanced currents have become highly K selective [99,100].…”

Influence of the N Terminus on the Biophysical Properties and Pharmacology of TREK1 Potassium Channels

“…Thomas et al (2008) describe the N terminus truncated form of TREK1 (TREK1DN) as being regionally and developmentally regulated in a number of different regions of the rat central nervous system. Both forms are expressed in COS-7 cells (Thomas et al, 2008) but predominantly the long form is found in human embryonic kidney 293 cells (Ma et al, 2011). For the related K2P channel TREK2, which also undergoes ATI (Simkin et al, 2008), the degree of ATI has been shown to be tissue specific (Staudacher et al, 2011) through differential regulation of mRNA translation.…”

“…TREK1DN has been described as a constitutively "nonconductive" variant, with a "collapsed selectivity filter" (Ma et al, 2011) that is permeable to sodium under normal physiologic conditions, leading to membrane depolarization when it is active in neurons (Thomas et al, 2008). Furthermore, TREK1DN has been shown to copurify with the longer form (Thomas et al, 2008), so heteromers may also exist.…”

[Ca2+]i-induced augmentation of the inward rectifier potassium current (IK1) in canine and human ventricular myocardium

“…[5]. However, we have very limited information about its possible effect on background leak channels [26], or on cardiac Ca 2+ activated K + channels. Therefore, we should not assign the significant Ca 2+ sensitivity of I K1 to a single channel type.…”