Gotowa bibliografia na temat „HTREK-1 Potassium Channel”

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2–15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and Nω-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

Abstract Background and purpose Mechanosensitive hTREK-1 (hK2P2.1) two-pore-domain potassium channels give rise to background currents that control resting membrane potential in excitable tissue. Recently TREK-1 currents have been linked to regulation of cardiac rhythm as well as hypertrophy and fibrosis. Even though pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, less is known about its posttranslational modifications. This study aims to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Experimental approach Following pharmacological inhibition of N glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-233T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. Key results TREK-1 channels subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrate that non-glycosylated hTREK-1 channels subunits are able to reach the cell surface in general, but seemingly with reduced efficiency. Conclusion and implications hTREK-1 are glycoproteins and N glycosylation at positions 110 and 134 is involved in channel surface trafficking. These findings extend our view on regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how glycosylation deficiency disorders may promote arrhythmogenesis.

TREK-1 is a two-pore domain potassium channel that contributes to maintenance of the resting membrane potential of a cell. TREK-1 is involved in several physiological and pathophysiological conditions like nociception, anaesthesia, epilepsy, ischemia and depression. Activity of TREK-1 is modulated by a number of physical and chemical stimuli including the activation of G-protein coupled receptors by several neurotransmitters and hormones. An important modulator of neuronal activity and function is 17β-estradiol, which by acting through its classical receptors ERα and ERβ, can bring about genomic changes in the cell. 17β-Estradiol can also act through membrane receptors like the G-protein coupled estrogen receptor (GPER) and activate intracellular signaling pathways. Several neuroprotective effects of 17β-estradiol in epilepsy, ischemia and diseases like Alzheimer‟s and Parkinson‟s is mediated through activation of GPER. 17β-Estradiol is also known to modulate the activity of several ion channels in a non-genomic manner, thus, regulating the neuronal function. Of the different membrane ionic channels, the leak potassium channel TREK-1 is implicated in neuroprotection since their activation hyperpolarizes the membrane of neurons and astrocytes and reduces neuronal excitability. However, it is not known whether 17β-estradiol can physiologically modulate the activity of TREK-1 channels and use this as an additional mechanism to mediate neuroprotection. In the present study, using single-channel cell-attached patch-clamp electrophysiology in HEK293 cells, we show that 17β-estradiol increases the activity of hTREK-1 by an hGPER-dependent mechanism. The probability of opening of the hTREK-1 channel increased rapidly and irreversibly on application of 17β-estradiol, not directly but only in the presence of hGPER. The potentiation of hTREK-1 activity by 17β-estradiol was mimicked by hGPER agonist and inhibited by hGPER antagonist, supporting the hGPER-dependence of 17β-estradiol action. Pharmacological studies demonstrated that the hGPER-mediated potentiation of hTREK-1 by 17β-estradiol occurred in a pertussis toxin-sensitive manner, mediated by the Gβγ subunits. Raising the intracellular cAMP levels reversed the potentiation of hTREK-1 induced by 17β-estradiol suggesting the inhibition of cAMP production in the hGPER-mediated increase of hTREK-1 activity. The hGPER-dependent rise in hTREK-1 activity induced by 17β-estradiol was occluded by inhibition of PKA which indicated that 17β-estradiol action involves inhibition of PKA. The serines at position 315 and 348 in the C-terminal domain of hTREK-1 are involved in phosphorylation-mediated inhibition of channel activity as known from earlier studies. Mutational studies with S315 and S348 suggested that S348 was the target site for dephosphorylation and potentiation of hTREK-1 by hGPER-mediated action of 17β-estradiol. 17β-Estradiol-induced potentiation of hTREK-1 was abolished on inhibition of serine/threonine phosphatases suggesting the requirement of serine/threonine phosphatases for the action of 17β-estradiol. Thus, the inhibition of PKA acts jointly with the activation of serine/threonine phosphatases to dephosphorylate S348 in the C-terminal domain of hTREK-1 leading to an increase in its activity. It was known from previous literature that TREK-1 and 17β-estradiol play important roles in neuroprotection. However, the effect of 17β-estradiol on the TREK-1 channel was not explored. The study undertaken as part of this thesis provides the link between 17β-estradiol and TREK-1 activity, giving an insight into a plausible mechanism underlying the several neuroprotective roles of 17β-estradiol.