Péter Enyedi

Molecular Background of Leak K+ Currents: Two-Pore Domain Potassium Channels

Two-pore domain K(+) (K(2P)) channels give rise to leak (also called background) K(+) currents. The well-known role of background K(+) currents is to stabilize the negative resting membrane potential and counterbalance depolarization. However, it has become apparent in the past decade (during the detailed examination of the cloned and corresponding native K(2P) channel types) that this primary hyperpolarizing action is not performed passively. The K(2P) channels are regulated by a wide variety of voltage-independent factors. Basic physicochemical parameters (e.g., pH, temperature, membrane stretch) and also several intracellular signaling pathways substantially and specifically modulate the different members of the six K(2P) channel subfamilies (TWIK, TREK, TASK, TALK, THIK, and TRESK). The deep implication in diverse physiological processes, the circumscribed expression pattern of the different channels, and the interesting pharmacological profile brought the K(2P) channel family into the spotlight. In this review, we focus on the physiological roles of K(2P) channels in the most extensively investigated cell types, with special emphasis on the molecular mechanisms of channel regulation.

Formation of Functional Heterodimers between the TASK-1 and TASK-3 Two-pore Domain Potassium Channel Subunits

The potassium channels in the two-pore domain family are widely expressed and regulate the excitability of neurons and other excitable cells. These channels have been shown to function as dimers, but heteromerization between the various channel subunits has not yet been reported. Here we demonstrate that two members of the TASK subfamily of potassium channels, TASK-1 and TASK-3, can form functional heterodimers when expressed in Xenopus laevis oocytes. To recognize the two TASK channel types, we took advantage of the higher sensitivity of TASK-1 over TASK-3 to physiological pH changes and the discriminating sensitivity of TASK-3 to the cationic dye ruthenium red. These features were clearly observed when the channels were expressed individually. However, when TASK-1 and TASK-3 were expressed together, the resulting current showed intermediate pH sensitivity and ruthenium red insensitivity (characteristic of TASK-1), indicating the formation of TASK-1/TASK-3 heterodimers. Expression of a tandem construct in which TASK-3 and TASK-1 were linked together yielded currents with features very similar to those observed when coexpressing the two channels. The tandem construct also responded to AT1a angiotensin II receptor stimulation with an inhibition that was weaker than the inhibition of homodimeric TASK-1 and greater than that shown by TASK-3. Expression of epitope-tagged channels in mammalian cells showed their primary presence in the plasma membrane consistent with their function in this location. Heteromerization of two-pore domain potassium channels may provide a greater functional diversity and additional means by which they can be regulated in their native tissues.

Targeting of Calcineurin to an NFAT-like Docking Site Is Required for the Calcium-dependent Activation of the Background K+ Channel, TRESK

The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT.

The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

Generation and role of calcium signal in adrenal glomerulosa cells

CONTENTS PAGE

Polyphosphoinositide metabolism in adrenal glomerulosa cells

We examined the effect of angiotensin II, a calcium-mobilizing hormone on polyphosphoinositide metabolism in isolated rat adrenal glomerulosa cells. In cells preloaded with [32P]phosphate or with [3H]inositol, stimulation with angiotensin resulted in an approx. 40% reduction in the radioactivity of triphosphoinositide (PtdIns4,5P2) within 15 s. Only a slight increase in radioactivity was observed in the subsequent 30 min. Changes in labelling of diphosphoinositide (PtdIns4P) showed similar kinetics. Incorporation studies also showed a reduction in the pool size of [32P]PtdIns4P and [32P]PtdIns4,5P2 in response to angiotensin. Production of inositol phosphates in the absence or presence of lithium, a cation-inhibiting myo-inositol-1-phosphatase, was examined in cells preloaded with [3H]inositol. The results indicate that the production rate of inositol tris- and bisphosphate shows a manifold increase in the first seconds of stimulation and remains enhanced for at least several minutes. The present data suggest that the rate of resynthesis of polyphosphoinositides also increases shortly after the activation of PtdIns4,5P2 phosphodiesterase. Corticotropin, a hormone acting via cyclic AMP, neither affected polyphosphoinositide metabolism nor modified the action of angiotensin II.

Role of Nox2 in elimination of microorganisms.

NADPH oxidase of the phagocytic cells (Nox2) transfers electrons from cytosolic NADPH to molecular oxygen in the extracellular or intraphagosomal space. The produced superoxide anion (O2·−) provides the source for formation of all toxic oxygen derivatives, but continuous O2·− generation depends on adequate charge compensation. The vital role of Nox2 in efficient elimination of microorganisms is clearly indicated by human pathology as insufficient activity of the enzyme results in severe, recurrent bacterial infections, the typical symptoms of chronic granulomatous disease. The goals of this contribution are to provide critical review of the Nox2-dependent cellular processes that potentially contribute to bacterial killing and degradation and to indicate possible targets of pharmacological interventions.

Phosphorylation-dependent Binding of 14-3-3 Proteins Controls TRESK Regulation

The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3η with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3η accelerated the return of the K+ current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3η was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The γ and η isoforms of 14-3-3 controlled TRESK regulation, whereas the β, ζ, ϵ, σ, and τ isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3η. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K+ channel family.

Effects of lithium on angiotensin-stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship

Turnover of 32P‐labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P] phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin‐stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.

TRESK: the lone ranger of two-pore domain potassium channels.

TRESK (TWIK-related spinal cord K(+) channel, KCNK18) belongs to the two-pore domain (K2P) background (leak) potassium channel family. Unlike other K2P channels, TRESK is activated by the calcium signal in heterologous expression systems. The activation is mediated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. TRESK is abundantly expressed in dorsal root and trigeminal ganglia. The active ingredient of Sichuan pepper, sanshool, has been suggested to evoke tingling paresthesia by inhibiting the channel in a mechanoreceptor subpopulation of sensory neurons. Recently, dominant-negative mutation of human TRESK was found to be linked to migraine with aura in a large pedigree. It is hoped that future TRESK agonists may prevent or ameliorate the debilitating symptoms of migraine. It will be interesting to see whether the calcineurin-activated K(+) channel maintains normal excitability in the cerebral cortex thereby arresting cortical spreading depression (CSD), or prevents migraine attack only in the trigeminovascular (TGVS) system.