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Membrane phosphatidylinositol- 4,5-bisphosphate (PIP2) is critical for the function of many transient receptor potential (TRP) ion channels. The role of PIP2 in TRPA1 function is not well known. The effect of PIP2 on TRPA1 was investigated by direct application of PIP2 and by using polylysine and PIP2 antibody that sequester PIP2. In inside-out patches from HeLa cells expressing mouse TRPA1, polytriphosphate (PPPi) was added to the bath solution to keep TRPA1 sensitive to allyl isothiocyanate (AITC; mustard oil). Direct application of PIP2 (10 µM) to inside-out patches did not activate TRPA1, but AITC and Δ9-tetrahydrocannabinol (THC) produced strong activation. In inside-out patches in which TRPA1 was first activated with AITC (in the presence of PPPi), further addition of PIP2 produced a concentration-dependent inhibition of TRPA1 [agonist concentration producing half-maximal activity (K1/2), 2.8 µM]. Consistent with the inhibition of TRPA1 by PIP2, AITC activated a large whole cell current when polylysine or PIP2 antibody was added to the pipette but a markedly diminished current when PIP2 was added to the pipette. In inside-out patches with PPPi in the bath solution, application of PIP2 antibody or polylysine caused activation of TRPA1, and this was blocked by PIP2. However, TRPA1 was not activated by polylysine and PIP2 antibody under whole cell conditions, suggesting a more complex regulation of TRPA1 by PIP2 in intact cells. These results show that PIP2 inhibits TRPA1 and reduces the sensitivity of TRPA1 to AITC. [ABSTRACT FROM AUTHOR]

Presents a study that examined how proteases, changes in intracellular pH, and different anions affect G protein-mediated activation and ATP-induced stimulation of KACh channel. Behavior of KACh with respect to G protein-induced activation; Methodology; Results of the study.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca 2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca 2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pH o ) on the kinetics of Ca 2+ oscillations. A decrease in pH o from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca 2+ oscillations. These effects of acidosis on Ca 2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K + channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca 2+ oscillations was inhibited by nifedipine (1 µM; L-type Ca v inhibitor) and by TTA-P2 (20 µM; T-type Ca v inhibitor). Mild inhibition of TASK activity by N -[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3- d ]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca 2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pH o 7.3) and mild hypoxia (∼5%O 2 ) produced similar levels of changes in the kinetics of Ca 2+ oscillations. Block of tetraethylammonium (TEA)-sensitive K v channels did not affect acid-induced increase in Ca 2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca 2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pH o .

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca 2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca 2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca 2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca 2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca 2+ entry (SOCE) using 2-APB abolished Ca 2+ oscillations. Inhibition of endoplasmic reticulum Ca 2+ -ATPase (SERCA) using thapsigargin abolished Ca 2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca 2+ oscillations. Inhibitors of L- and T-type Ca 2+ channels (Ca v ; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca 2+ oscillations. Thus, Ca 2+ oscillations observed in normoxia were caused by cyclical Ca 2+ fluxes at the ER, which was supported by Ca 2+ influx via Ca 2+ channels. Hypoxia (2-5% O 2 ) increased the frequency and amplitude of Ca 2+ oscillations, and Ca v inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca 2+ oscillations represent the basic Ca 2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.

TREK-2 is a member of the two-pore domain K(+) channel family and provides part of the background K(+) current in many types of cells. Neurotransmitters that act on receptors coupled to G(q) strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M(3) receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3-thiotriphosphate) (10 microM), Ca(2+) (5 microM), or diacylglycerol (DAG; 10 microM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 microM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 microM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via M(3) receptor occurs primarily via PKC-mediated phosphorylation.

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K(+) (K(2P)) channels that behave as background K(+) channels. To identify functional background K(+) channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K(+) channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K(+) channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K(2P) channel family. The molecular identity of the three other K(+) channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K(2P) channels, the TRESK-like (14 pS) K(+) channel was most active at 24 degrees C. At 37 degrees C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K(+) current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K(2P) channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K(+) conductance of DRG neurons at 37 degrees C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons.

TALK-1a, originally isolated from human pancreas, is a member of the tandem-pore K+ channel family. We identified and characterized three novel splice variants of TALK-1 from human pancreas. The cDNAs of TALK-1b, TALK-1c, and TALK-1d encode putative proteins of 294, 322, and 262 amino acids, respectively. TALK-1a and TALK-1b possessed all four transmembrane segments, whereas TALK-1c and TALK-1d lacked the fourth transmembrane domain because of deletion of exon 5. Northern blot analysis showed that among the 15 tissues examined, TALK-1 was expressed mainly in the pancreas. TALK-1a and TALK-1b, but not TALK-1c and TALK-1d, could be functionally expressed in COS-7 cells. Like TALK-1a, TALK-1b was a K+-selective channel that was active at rest. Single-channel openings of TALK-1a and TALK-1b were extremely brief such that the mean open time was <0.2 ms. In symmetrical 150 mM KCl, the apparent single-channel conductances of TALK-1a and TALK-1b were 23 +/- 3 and 21 +/- 2 pS at -60 mV and 11 +/- 2 and 10 +/- 2 pS at +60 mV, respectively. TALK-1b whole cell current was inhibited 31% by 1 mM Ba2+ and 71% by 1 mM quinidine but was not affected by 1 mM tetraethylammonium, 1 mM Cs+, and 100 microM 4-aminopyridine. Similar to TALK-1a, TALK-1b was sensitive to changes in external pH. Acid conditions inhibited and alkaline conditions activated TALK-1a and TALK-1b, with a K1/2 at pH 7.16 and 7.21, respectively. These results indicate that at least two functional TALK-1 variants are present and may serve as background K+ currents in certain cells of the human pancreas.