Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K+ channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3

Trafficking of neuronal two pore domain potassium channels.

The activity of two pore domain potassium (K2P) channels regulates neuronal excitability and cell firing. Post-translational regulation of K2P channel trafficking to the membrane controls the number of functional channels at the neuronal membrane affecting the functional properties of neurons. In this review, we describe the general features of K channel trafficking from the endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus then focus on established regulatory mechanisms for K2P channel trafficking. We describe the regulation of trafficking of TASK channels from the ER or their retention within the ER and consider the competing hypotheses for the roles of the chaperone proteins 14-3-3, COP1 and p11 in these processes and where these proteins bind to TASK channels. We also describe the localisation of TREK channels to particular regions of the neuronal membrane and the involvement of the TREK channel binding partners AKAP150 and Mtap2 in this localisation. We describe the roles of other K2P channel binding partners including Arf6, EFA6 and SUMO for TWIK1 channels and Vpu for TASK1 channels. Finally, we consider the potential importance of K2P channel trafficking in a number of disease states such as neuropathic pain and cancer and the protection of neurons from ischemic damage. We suggest that a better understanding of the mechanisms and regulations that underpin the trafficking of K2P channels to the plasma membrane and to localised regions therein may considerably enhance the probability of future therapeutic advances in these areas

Arrhenius plots presenting the effect of Ca²⁺ on the rate of cAMP-triggered exocytosis.

<p>Absolute values of Δ<i>C</i>/Δ<i>t</i> at t = 2 min plotted against the inverse of temperature (T) in the absence <b>(A)</b> and presence <b>(B)</b> of cytosolic Ca²⁺. The curve represents a linear least-squares fit to the equation lnk = lnA—E_A/RT. E_A is the energy of activation, k is the change in Δ<i>C</i>/Δ<i>t</i> upon a temperature change, A is the pre-exponential factor. R represents the gas constant and T the absolute temperature as usual. The negative slope of the curve gives activation energies of 5.7 kJ mol^-1 (A) and 53 kJ mol^-1 (B).</p

Undifferentiated and mature 3T3-L1 adipocytes as well as demonstration of analysis performance.

<p><b>A</b> Example of 3T3-L1 adipocytes in the fibroblast-like state before differentiation (left) as well as when differentiated into mature adipocytes (right). <b>B</b> Representative capacitance recording with Δ<i>C</i>_m (delta membrane capacitance) plotted against time showing how analyses were carried out. Δ<i>C</i>/Δ<i>t</i> was measured at the time intervals indicated by the dotted lines by fitting straight functions to the data points (red lines superimposed on the black capacitance trace). Scale bar = 50μm.</p

Ca²⁺-augmented adiponectin secretion is abolished by cooling while secretion stimulated by cAMP alone is unaffected.

<p><b>A</b> Adiponectin secretion as fold-increase compared to control stimulated by 8-Br-cAMP (1 mM) alone or in combination with ionomycin (1 μM) during 30 min incubations at 23°C (blue) or 32°C (red). <b>B</b> As in <b>(A)</b> but using forskolin (10 μM) and IBMX (200 μM; forsk/IBMX) as a stimulator. Data are mean values ± S.E.M. of 11 experiments at each temperature in (A) and 7 (23°C) and 8 (32°C) in (B). <i>*P<0</i>.<i>05; **P<0</i>.<i>01</i>; <i>**P<0</i>.<i>001</i> vs. control at corresponding temperature. †<i>P<0</i>.<i>01</i> vs. 8-Br-cAMP or forsk/IBMX alone at 32°C; ǂ <i>P<0</i>.<i>05</i> vs. 8-Br-cAMP + ionomycin or forsk/IBMX + ionomycin at room temperature. The data in (B) at 32°C are the same as in Fig. 7B in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119530#pone.0119530.ref005" target="_blank">5</a>].</p

Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca²⁺]_i.

<p>Example traces of [Ca²⁺]_i responses upon extracellular application of 1 μM ionomycin <b>(A)</b> or 1 mM 8-Br-cAMP <b>(C)</b>. <b>B</b> Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.</p

Proposed model of cooling effects on white adipocyte exocytosis.

<p>White adipocytes release adiponectin containing vesicles belonging to two functionally distinct populations. cAMP stimulates release of vesicles residing in a readily releasable pool in a temperature-independent manner. The Ca²⁺-dependent augmentation of secretion is reduced/abolished by cooling. Intracellular ATP is necessary for the Ca²⁺ effect. See text for more details.</p

Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis

Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca2+ and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca2+-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof