Cross‐talk between the mechano‐gated K 2P channel TREK‐1 and the actin cytoskeleton

Twik-related K + channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K + channel (TRAAK) form the TREK subfamily of two-pore-domain K + (K 2P ) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K 2P channels.potassium channels | single-molecule fluorescence | leak current | combinatorial diversity | heteromerization

“…S2 A and B). Consistent with previous studies (18,25), we found that HA-TASK3 pulled down GFP-TASK3 or GFP-TASK1 with similar efficiency (Fig. S2C).…”

Section: Resultssupporting

confidence: 92%

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels

Levitz

Royal

Comoglio

et al. 2016

“…3 B, D, and E). This observation supports the idea that desensitization does not arise from the cytoskeleton, because that is expected to be similar after transfection with different channels [although see our previous report showing that cytoskeleton structure can be coupled to specific K 2P channel expression (25)]. The average P 1/2 value determined by fitting pressure-effect curves with a Boltzmann function (see Fig.…”

Section: Resultssupporting

confidence: 87%

“…The effect of patch excision is actually mimicked by latrunculin A treatment in the cellattached configuration (see ref. 25 and Fig. 6B).…”

Section: Resultsmentioning

confidence: 75%

See 1 more Smart Citation

Desensitization of mechano-gated K _2P channels

Honoré

Patel

Chemin

et al. 2006

Self Cite

126

115

The neuronal mechano-gated K2P channels TREK-1 and TRAAK show pronounced desensitization within 100 ms of membrane stretch. Desensitization persists in the presence of cytoskeleton disrupting agents, upon patch excision, and when channels are expressed in membrane blebs. Mechanosensitive currents evoked with a variety of complex stimulus protocols were globally fit to a four-state cyclic kinetic model in detailed balance, without the need to introduce adaptation of the stimulus. However, we show that patch stress can be a complex function of time and stimulation history. The kinetic model couples desensitization to activation, so that gentle conditioning stimuli do not cause desensitization. Prestressing the channels with pressure, amphipaths, intracellular acidosis, or the E306A mutation reduces the peak-to-steady-state ratio by changing the preexponential terms of the rate constants, increasing the steady-state current amplitude. The mechanical responsivity can be accounted for by a change of in-plane area of Ϸ2 nm 2 between the closed and open conformations. Desensitization and its regulation by chemical messengers is predicted to condition the physiological role of K2P channels.M ost receptors, including ligand-gated ion channels, desensitize with continuous stimulation. Desensitization is a reversible, use-dependent, form of signal plasticity critical for maintaining the dynamic sensitivity over a wide dynamic range. Desensitization also protects cells from inappropriate persistent activation. Many mechano-gated ion channels desensitize (1-5). There are two basic mechanisms that account for desensitization: adaptation and inactivation. Adaptation refers to the uncoupling of the stimulus from the channel, whereas inactivation refers to a block of the permeation path. In hair cells of the inner ear, deflection opens a mechano-gated channel at the tip of the stereocilium leading to depolarization (6). A fast adaptation mechanism (0.3-5 ms) is attributed to the binding of calcium near the channel after permeation through the open ionic pore (6), i.e., Ca 2ϩ block. A slower adaptation (10-100 ms) occurs when the calcium-dependent molecular motor myosin-1c, which pulls on the channels, slips down the actin cytoskeleton reducing the force applied to the channel (6).Rapid desensitization also occurs in mechanosensitive channels expressed in nonspecialized cells. For instance, the stretchactivated cation channel of Xenopus oocyte opens transiently in response to a step change in patch pressure (5). The decrease in current has been attributed to relaxation of the mechanical stimulus, although as shown in this work, that interpretation may not be a good discriminator. Desensitization of the oocyte channel is fragile and may disappear when patches are repetitively stimulated (5). This fragility was attributed to decoupling of the bilayer from the cytoskeleton (5).Stretch-activated K ϩ channels are present in neurons of both the central and the peripheral nervous systems (7-9), and they also have been described in nonne...

“…Similar observations have also been made on mechanosensitive channels of known molecular identity. For example, treatment with latrunculin, another such agent, greatly enhances the suction-induced current through TREK-1-expressed in COS cells (85). Mutating β-spectrin, which links the membrane to the cytoskeleton, reduces the force needed to pull membrane tether from cultured touch-receptor neurons of the worm with an atomic force microscope (86).…”

Section: Accessoriesmentioning

confidence: 99%

Feeling the hidden mechanical forces in lipid bilayer is an original sense

Anishkin

Loukin²,

Teng³

et al. 2014

249

216

Life's origin entails enclosing a compartment to hoard material, energy, and information. The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous domains. The self-assembled lipid bilayer comes with a set of properties including its strong anisotropic internal forces that are chemically or physically malleable. Added bilayer stretch can alter force vectors on embedded proteins to effect conformational change. The force-from-lipid principle was demonstrated 25 y ago when stretches opened purified Escherichia coli MscL channels reconstituted into artificial bilayers. This reductionistic exercise has rigorously been recapitulated recently with two vertebrate mechanosensitive K + channels (TREK1 and TRAAK). Membrane stretches have also been known to activate various voltage-, ligand-, or Ca 2+ -gated channels. Careful analyses showed that Kv, the canonical voltage-gated channel, is in fact exquisitely sensitive even to very small tension. In an unexpected context, the canonical transient-receptor-potential channels in the Drosophila eye, long presumed to open by ligand binding, is apparently opened by membrane force due to PIP 2 hydrolysis-induced changes in bilayer strain. Being the intimate medium, lipids govern membrane proteins by physics as well as chemistry. This principle should not be a surprise because it parallels water's paramount role in the structure and function of soluble proteins. Today, overt or covert mechanical forces govern cell biological processes and produce sensations. At the genesis, a bilayer's response to osmotic force is likely among the first senses to deal with the capricious primordial sea. mechanosensitivity | force sensing | channel gating | bilayer mechanics | touch