The Phosphodiesterase Inhibitor IBMX Blocks the Potassium Channel THIK-1 from the Extracellular Side.

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.

Membrane proteins as 14-3-3 clients in functional regulation and intracellular transport.

14-3-3 proteins regulate the function and subcellular sorting of membrane proteins. Often, 14-3-3 binding to client proteins requires phosphorylation of the client, but the relevant kinase is unknown in most cases. We summarize current progress in identifying kinases that target membrane proteins with 14-3-3 binding sites and discuss the molecular mechanisms of 14-3-3 action. One of the kinases involved is Akt/PKB, which has recently been shown to activate the 14-3-3-dependent switch in a number of client membrane proteins.

Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages.

STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion.

Enantioselective actions of bupivacaine and ropivacaine on coronary vascular resistance at cardiotoxic concentrations.

The main concern with the use of the long-acting local anesthetics bupivacaine and ropivacaine is inadvertent IV injection, which exposes the heart to toxic drug concentrations. We tested the hypothesis that these chiral anesthetics exert enantioselective actions on coronary vascular tone, the regulation of which does not involve voltage-gated Na(+) channels. Coronary perfusion pressure (CPP) was continuously measured in isolated hearts perfused via the aorta at a constant flow rate. This method provides a sensitive assay of coronary vascular resistance in the intact heart. In parallel experiments, we examined the effects of bupivacaine and ropivacaine on intracellular [Ca(2+)] in coronary endothelial cells. In addition, the effect of bupivacaine on mitochondrial membrane potential was assessed using isolated ventricular myocytes. Racemic bupivacaine and R(+)-bupivacaine produced similar dose-dependent decreases in CPP. However, S(-)-bupivacaine, S(-)-ropivacaine and R(+)-ropivacaine increased CPP. In contrast to adenosine triphosphate, neither racemic bupivacaine nor S(-)-ropivacaine changed endothelial intracellular [Ca(2+)], suggesting that these clinically used drugs do not modulate endothelial nitric oxide synthase. We also showed that the putative uncoupler bupivacaine did not depolarize mitochondria in intact ventricular myocytes. In conclusion, the long-acting local anesthetics have enantioselective actions on coronary resistance vessels. Racemic bupivacaine and R(+)-bupivacaine are coronary vasodilators, whereas S(-)-bupivacaine, S(-)-ropivacaine and, to a lesser extent, R(+)-ropivacaine all induce coronary vasoconstriction.

Extracellular ATP induces oscillations of intracellular Ca2+ and membrane potential and promotes transcription of IL-6 in macrophages.

The effects of low concentrations of extracellular ATP on cytosolic Ca(2+), membrane potential, and transcription of IL-6 were studied in monocyte-derived human macrophages. During inflammation or infection many cells secrete ATP. We show here that application of 10 microM ATP or 10 microM UTP induces oscillations in cytosolic Ca(2+) with a frequency of approximately 12 min(-1) and oscillations in membrane potential. RT-PCR analysis showed expression of P2Y(1), P2Y(2), P2Y(11), P2X(1), P2X(4), and P2X(7) receptors, large-conductance (KCNMA1 and KCNMB1-4), and intermediate-conductance (KCNN4) Ca(2+)-activated K(+) channels. The Ca(2+)oscillations were unchanged after removal of extracellular Ca(2+), indicating that they were mainly due to movements of Ca(2+) between intracellular compartments. Comparison of the effects of different nucleotides suggests that the Ca(2+) oscillations were elicited by activation of P2Y(2) receptors coupled to phospholipase C. Patch-clamp experiments showed that ATP induced a transient depolarization, probably mediated by activation of P2X(4) receptors, followed by membrane potential oscillations due to opening of Ca(2+)-activated K(+) channels. We also found that 10 microM ATP gamma S increased transcription of IL-6 approximately 40-fold within 2 h. This effect was abolished by blockade of P2Y receptors with 100 microM suramin. Our results suggest that ATP released from inflamed, damaged, or metabolically impaired cells represents a "danger signal" that plays a major role in activating the innate immune system.

Beta-oxidation of 5-hydroxydecanoate, a putative blocker of mitochondrial ATP-sensitive potassium channels.

5-Hydroxydecanoate (5-HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5-HD is thought to inhibit specifically the putative mitochondrial ATP-sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5-HD is a substrate for acyl-CoA synthetase, the mitochondrial enzyme which 'activates' fatty acids. Here, we tested whether activated 5-HD, 5-hydroxydecanoyl-CoA (5-HD-CoA), is a substrate for medium-chain acyl-CoA dehydrogenase (MCAD), the committed step of the mitochondrial beta-oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5-HD-CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5-HD-CoA was a substrate for purified human liver MCAD with a Km of 12.8 +/- 0.6 microM and a kcat of 14.1 s-1. For comparison, with decanoyl-CoA (Km approximately 3 microM) as substrate, kcat was 6.4 s-1. 5-HD-CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5-hydroxydecenoyl-CoA (5-HD-enoyl-CoA), was a substrate for enoyl-CoA hydratase, the second enzyme of the beta-oxidation pathway. Similar to decenoyl-CoA, purified 5-HD-enoyl-CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5-HD is metabolised at least as far as the third enzyme of the beta-oxidation pathway. Our results open the possibility that beta-oxidation of 5-HD or metabolic intermediates of 5-HD may be responsible for the inhibitory effects of 5-HD on preconditioning of the heart.

K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart.

Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Here we describe K(ATP) channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 microM) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective K(ATP) channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial K(ATP) channels. Furthermore, application of either diazoxide (100 microM) or pinacidil (100 microM) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was 'activated' via acyl-CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (beta-oxidation) and/or exerting some other cellular action. In conclusion, we have identified K(ATP) channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial K(ATP) channels. We propose that pharmacological preconditioning may be related to partial inhibition of respiratory chain complexes.