Voltage-sensing domains (VSDs) confer voltage dependence on effector domains of membrane proteins. Ion channels use four VSDs to control a gate in the pore domain, but in the recently discovered phosphatase Ci-VSP, the number of subunits has been unknown. Using single-molecule microscopy to count subunits and voltage clamp fluorometry to detect structural dynamics, we found Ci-VSP to be a monomer, which operates independently, but nevertheless undergoes multiple voltage-dependent conformational transitions.

In the voltage sensing phosphatase, Ci-VSP, a voltage sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we find that the inter-domain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays show that the linker is also needed for activity in the isolated PD. We identify a late step of VSD motion in the full-length protein that depends on the linker. Strikingly, this VSD motion is found to require PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme.

TREK channels produce background currents that regulate cell excitability. These channels are sensitive to a wide variety of stimuli including polyunsaturated fatty acids (PUFAs), phospholipids, mechanical stretch, and intracellular acidification. They are inhibited by neurotransmitters, hormones, and pharmacological agents such as the antidepressant fluoxetine. TREK1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics, altered perception of pain, and a depression-resistant phenotype. Here, we investigate TREK1 regulation by Gq-coupled receptors (GqPCR) and phospholipids. Several reports indicate that the C-terminal domain of TREK1 is a key regulatory domain. We developed a fluorescent-based technique that monitors the plasma membrane association of the C terminus of TREK1 in real time. Our fluorescence and functional experiments link the modulation of TREK1 channel function by internal pH, phospholipid, and GqPCRs to TREK1-C-terminal domain association to the plasma membrane, where increased association results in greater activity. In keeping with this relation, inhibition of TREK1 current by fluoxetine is found to be accompanied by dissociation of the C-terminal domain from the membrane.T REK1 is a two-pore-domain K + (K 2P ) channel that produces a nearly time-and voltage-independent background current. This current drives the membrane potential toward the K + equilibrium potential and thus affects input resistance. TREK1 displays low basal activity when expressed alone (1) but can be strongly stimulated by temperature (2), mechanical stretch (3), external alkalization (4), intracellular acidification (5), polyunsaturated fatty acids (PUFAs) (6), lysophospholipids (7), phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] (8, 9), and pharmacological agents such as volatile anesthetics (10) and riluzole (11). TREK1 is inhibited by neurotransmitters and hormones that activate Gq and Gs pathways (3,(12)(13)(14) and pharmacological agents such as the antidepressant drug fluoxetine (15). TREK1 gene inactivation produces mice with decreased sensitivity to volatile anesthetics, impaired PUFA-mediated neuroprotection (16), and altered perception of pain (17). These mice also display a depression-resistant phenotype (18). This phenotype is in agreement with the sensitivity of TREK1 to fluoxetine, a widely used antidepressant drug.In the last decade, much effort has been made to elucidate the gating mechanism of the TREK1 channel. The cytosolic carboxylterminal domain of TREK1 that follows its fourth transmembrane domain (post-M4) has been implicated in its function and regulation. A glutamate residue, E306, has been shown to be a key element for stimulation by intracellular acidification (19) and a cluster of basic residues in the same region has been shown to be involved in TREK1 regulation by phospholipid (8). However, the mechanism of regulation of the channel by Gq-coupled receptors (GqPCR) and by pharmacological agents such as fluoxetine remains unclear.I...

Potassium channels are among the core functional elements of life because they underpin essential cellular functions including excitability, homeostasis, and secretion. We present here a series of multivalent calix [

With the discovery of the Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), voltage sensing domains (VSDs) have moved beyond the exclusive realm of ion channels. By combining a VSD with a lipid phosphatase domain, Ci-VSP is the first enzyme known to be directly regulated by voltage. Interestingly, Ci-VSP is also the first protein with a monomeric VSD. Yet, a monomeric VSD still exhibits complex protein motions indicating that other factors beyond the oligomerization state of the domain play a part. We have applied two electrode voltage clamp electrophysiology to probe phosphatase function as well as voltage clamp fluorometry to probe the conformational changes involved in voltage sensing and the propagation of that signal to the phosphatase. The observed changes in fluorescence correlate with protein motions allowing any factors affecting the protein to be teased apart. These motions are influenced by the catalytic states of the phosphatase consistent with the expected coupling between the two domains. The linker between the voltage sensing domain and the phosphatase domain also influences these motions suggesting the linker may play an active role in protein regulation. By investigating Ci-VSP, we hope to gain a greater understanding of how the VSD functions.

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After publication, the authors found second-site mutations within two of the Ci-VSP constructs. The R253Q G214C and R254Q G214C constructs have been remade without the additional mutations and the data recollected. The data originally presented in Figure 1, Supplementary Figures 5 and 6 and Supplementary Table 1 have been updated to reflect the minor changes in the magnitude of the constructs' effects. The changes do not affect the conclusions of the study. The changes have been made in the HTML and PDF versions of the article.