Inside-out Configuration Research Articles

Heat and AITC activate green anole TRPA1 in a membrane-delimited manner

Transient receptor potential ankyrin 1 (TRPA1) is a member of the large TRP super family of ion channels and functions as a Ca(2+)-permeable nonselective cation channel that is activated by various noxious stimuli. TRPA1 was initially identified as a potential mediator of noxious cold stimuli in mammalian nociceptive sensory neurons, while TRPA1s from nonmammalian vertebrates (snakes, green anole lizards, and frogs) were recently reported to be activated by heat, but not cold stimulus. In this study, we examined detailed properties of the green anole TRPA1 channel (gaTRPA1) related to thermal and chemical stimulation in whole-cell and single-channel recordings. Heat activates gaTRPA1 with a temperature threshold for activation of 35.8°C, while heat together with allyl isothiocyanate (AITC), a chemical agonist, had synergistic effects on gaTRPA1 channel activation in that either the temperature threshold or activating AITC concentration was reduced in the presence of the other stimulus. Significant heat-evoked gaTRPA1 activation was observed in the presence but not absence of extracellular Ca(2+). gaTRPA1 channels were also activated by heat and AITC in excised membrane patches with an inside-out configuration. By comparing the kinetics of heat- and AITC-evoked single-channel currents, we defined similarities and differences of gaTRPA1 channel responses to heat and AITC. We observed similar current-voltage relationship and unitary amplitudes for heat- and AITC-evoked currents and found that heat-activated currents showed shorter durations of both open and closed times. Our results suggest that the gaTRPA1 channel is directly activated by heat and chemical stimuli.

The N-Terminus of Auxiliary β Subunit is Involved in the Modulation of Voltage Sensor BK Pore-Forming α Subunit

BK channels and their modulatory β subunits are found in most mammalian tissues ranging from brain to liver to smooth muscle, where they could be modulated by specific β subunits (β1-β4). Recently, we showed that auxiliary β1 and β2 subunits stabilize the BK voltage sensor in the active conformation, and that β3 has no effect on voltage sensor equilibrium (Contreras et al., 2012). Despite the fact that the BK phenotype produced by each of the β subunits has been well characterized, controversies exist regarding the molecular mechanisms by means of which these auxiliary subunits can modify the gating of BK channel. To identify the molecular determinants that confer to the β1 the ability to modulate the voltage sensor of BK channels, we constructed fifteen chimeras of β1/β2 and β1/β3 subunits by exchanging the N- and C-terminus, the transmembrane (TM) segments, and the extracellular loops, between them. The RNA of chimeric constructs were injected in Xenopus laevis oocites and gating currents were recorded in permeant ions free-solutions and in the absence of internal Ca2+ by patch clamp on inside-out configuration. Gating currents were elicited by steps of 1 ms to increasing voltages ranging from −90 to 350 mV in increments of 10 mV, and integrated during the onset of depolarizing pulses to generate charge versus voltage (Q/V) curves. Our results show that the effects of the β1 subunit on the resting-active equilibrium of the voltage sensor is determined by the N- and C-terminal with a major contribution of the N-terminus.

Regulation of TRPV1 by Phosphoinositides and other Negatively Charged Lipids

The capsaicin receptor Transient Receptor Potential Vanilloid 1 (TRPV1) is a polymodal, Ca2+-permeable ion channel essential for nociception. Like most members of the TRP family, it is known to be regulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Although the majority of PI(4,5)P2-regulated ion channels are activated by this phosphoinositide, TRPV1 was initially suggested to experience inhibition. Data from cellular systems support the idea that TRPV1 activity depends on the presence of phosphoinositides in the membrane. The purified TRPV1, on the other hand, was recently shown to be fully functional in artificial liposomes in the absence of phosphoinositides. We expressed the rat TRPV1 channel in Xenopus oocytes and performed patch-clamp experiments in the excised inside-out configuration. We found that in addition to PI(4,5)P2, several other negatively charged phospholipids, including phosphatidylglycerol, could also support TRPV1 activity in excised patches. When we incorporated TRPV1 into planar lipid bilayers consisting of neutral lipids, capsaicin-induced activity depended on PI(4,5)P2. We also found that TRPV1 activity in excised patches ran down upon excision and that perfusing the patch with MgATP restored channel activity. Inhibition of phosphatidylinositol 4-Kinases or enzymatic removal of phosphatidylinositol abolished this effect, suggesting that MgATP activated TRPV1 by generating endogenous phosphoinositides. We conclude that endogenous phosphoinositides are positive cofactors for TRPV1 activity. Our data highlight the importance of specificity in lipid regulation of ion channels, and may reconcile discordant data obtained in various experimental settings.

Functional expression of mechanosensitive two-pore domain potassium channel in human bladder carcinoma cells

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at ?80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current-voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

ASSA13-03-35 Establishment and Identification of Stable Cell Line HEK293/BKCA in Molecular and Electrophysiological Levels

<h3>Objective</h3> Large conductance Ca²⁺-sensitive K⁺ channels (BK_Ca) are involved in dysfunctional relaxation of artery in patients with hypertension and represent an attractive target for drug discovery. In this study, we aimed to establish the stable cell line HEK293/BK_Ca as a future in-vitro drug-screening platform. <h3>Methods</h3> HEK293 cell with stable expression of BK_Ca was constructed through transfecting the plasmid pcDNA3.1-BK_Ca. Molecular and electrophysiological characteristic of HEK293/BK_Ca were identified by molecular biology and patch clamp techniques. <h3>Results</h3> Semiquantitative RT-PCR and Western blot showed that BK_Ca was highly expressed in HEK293/BK_Ca while not in control cells. Using Immunofluorescence method, strong positive fluorescence signal for BK_Ca was observed specifically in membrane of BK_Ca cells. Single-channel patch clamp experiments showed that the open probability of BK_Ca in the cell was voltage and calcium dependent. In insideout configuration, The Am and <i>N</i>Po of single-channel in HEK293/BK_Ca gradually enhanced with the increased voltage (0, 10, 20, 30, 40, 50, 60 mV). Moreover, The Am and <i>N</i>Po also showed Increasing tendency with increased calcium concentration (0, 122, 187 nM) at a certain voltage. External applied IBTX (BK_Ca specific antagonist) led to complete inhibition in channel activity. <h3>Conclusions</h3> Taken together, these results indicated that HEK293/BK_Ca stable cell line was successfully constructed and BK_Ca channel expressed in HEK293 cells exhibited functional features similar to native BK_Ca channels. Which would serve as an efficient tool for future drug-screening targeted BK_Ca.

Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells.

Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

The Inhibition of Inwardly Rectifying K+ Channels by Memantine in Macrophages and Microglial Cells

Background/Aims: Memantine (MEM) can block N-methyl-D-aspartate receptors non-competitively and is recognized to exert anti-inflammatory action. Whether MEM and other related compounds produce any effects on K⁺ currents in macrophages and in microglial cells is largely unknown. In this study, we investigated the effects of MEM and other related compounds on inwardly rectifying K⁺ current (I_K(IR)) in RAW 264.7 macrophages and in BV2 microglial cells. Methods: Patch-clamp recordings under whole-cell, cell-attached or inside-out configuration were performed in standard patch-clamp technique. MEM suppressed the I_K(IR) amplitude in a concentration-dependent manner with an IC₅₀ value of 12 µM. Results: This agent significantly slowed the inactivation time rate of I_K(IR) evoked with membrane hyperpolarization. In cells dialyzed spermine (10 µM), MEM-mediated inhibition of I_K(IR) no longer existed. MEM-suppressed activity is associated with a decrease in the slow component of mean open time and an increase in mean closed time, despite no detectable change in single-channel conductance of inwardly rectifying K⁺ (Kir) channels. Under current-clamp conditions, the addition of MEM resulted in membrane depolarization of RAW 264.7 cells. Similarly, in BV2 microglial cells, addition of MEM suppressed I_K(IR) as well as depolarized the membrane. However, neither C6 astrocytic cells nor Jurkat T-lymphoces were noted to display I_K(IR). Conclusion: The block by MEM of Kir2.1 channels is thus one of the important mechanisms underlying its actions on the functional activities of either macrophages or microglial cells, if similar findings occur in vivo.

Deletion of the Amino-Terminus Uncovers an Inactivated State in EAG1 Channels

Ether-a-go-go (Eag) family channels are voltage-gated K+ channels that are important in cardiac and neural function and have a well-documented role in disease. One of eight members of the mammalian Eag family, EAG1 channels expression is confined to the central nervous system of the healthy adult. EAG1 channels are also expressed in cancer cells and are implicated in tumor progression and regulation of the cell cycle. Most members of the Eag channel family exhibit voltage-dependent inactivation, yet wild-type EAG1 channels exhibit only voltage-gated activation and deactivation, with no apparent inactivation. Here we report that deletion of the entire intracellular amino-terminal domain uncovers an inactivated channel gating state at depolarizing potentials. We characterized this inactivated state in excised patches from Xenopus oocytes expressing mutant EAG1 channels, and recorded their currents in the inside-out configuration of the patch clamp technique. Similar to wild-type channels, EAG1 with a deleted amino-terminus begins to activate around −80 mV, however, these channels also inactivate at potentials higher than −40 mV. The rate of inactivation becomes faster with increasingly depolarizing potentials. Additionally, the kinetics of the EAG1 channels with a deleted amino-terminus are quite slow. Other members of the Eag family of channels exhibit C-type inactivation, which results from collapse of the selectivity filter. To determine whether the inactivation found in this EAG1 mutant was also C-type, we mutated a pore-lining residue known to alter C-type inactivation in both Shaker and hERG1 channels. As a second approach, we asked whether triethylammonium applied to the external surface of the channel altered the rate of inactivation. Results from both experiments are inconsistent with C-type inactivation. Instead, we hypothesize that inactivation in the EAG1 channel lacking their amino-terminus is simply a reclosure of the activation gate.

Alterations in ventricular KATP channel properties during aging

Coronary heart disease remains the principle cause of mortality in the United States. During aging, the efficiency of the cardiovascular system is decreased and the aged heart is less tolerant to ischemic injury. ATP-sensitive K(+) (K(ATP)) channels protect the myocardium against ischemic damage. We investigated how aging affects cardiac K(ATP) channels in the Fischer 344 rat model. Expression of K(ATP) channel subunit mRNA and protein levels was unchanged in hearts from 26-month-old vs. 4-month-old rats. Interestingly, the mRNA expression of several other ion channels (> 80) was also largely unchanged, suggesting that posttranscriptional regulatory mechanisms occur during aging. The whole-cell K(ATP) channel current density was strongly diminished in ventricular myocytes from aged male rat hearts (also observed in aged C57BL/6 mouse myocytes). Experiments with isolated patches (inside-out configuration) demonstrated that the K(ATP) channel unitary conductance was unchanged, but that the inhibitory effect of cytosolic ATP on channel activity was enhanced in the aged heart. The mean patch current was diminished, consistent with the whole-cell data. We incorporated these findings into an empirical model of the K(ATP) channel and numerically simulated the effects of decreased cytosolic ATP levels on the human action potential. This analysis predicts lesser activation of K(ATP) channels by metabolic impairment in the aged heart and a diminished action potential shortening. This study provides insights into the changes in K(ATP) channels during aging and suggests that the protective role of these channels during ischemia is significantly compromised in the aged individual.

Trypanosoma brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

Voltage-gated sodium selective ion channel NaV1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. NaV1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon NaV1.5 to modulate activity by multiple mechanisms. This study examined whether NaV1.5 mechanosensitivity is modulated by local anesthetics. NaV1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V1/2a) and inactivation (V1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V1/2a but not V1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V1/2a. Lidocaine inhibited mechanosensitivity in NaV1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A NaV1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of NaV1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.

The inhibition by di(2-ethylhexyl)-phthalate of erg-mediated K+ current in pituitary tumor (GH3) cells

DEHP (bis(2-ethylhexyl)-phthalate) known to be an endocrine-disrupting chemical is a widely used phthalate. Little information regarding the effects of phthalate esters on ion currents is available. In this study, the effects of DEHP and other phthalate esters (DBEP: di(2-butoxyethyl)-phthalate and DMGP: di(2-methylglycol)-phthalate) on ion currents were investigated in pituitary GH₃ cells. Hyperpolarization-elicited K⁺ currents in GH3 cells bathed in high-K⁺, Ca²⁺-free solution were examined to evaluate the effects of DEHP, DBEP, and DMGP on the ether-a`-go-go-related-gene (erg) K⁺ current (IK(erg)). Addition of DEHP to GH₃ cells suppressed the amplitude of IK(erg) in a concentration-dependent manner with an IC₅₀ value of 16.3 μM. With a two-pulse protocol, addition of DEHP shifted the activation curve of IK(erg) to a depolarized potential by approximately 10 mV with no change in the rate of IK(erg) deactivation. This compound did not have any effects on delayed rectifier K⁺ current in GH₃ cells, while 4-aminopyridine-3-methanol (100 μM) suppressed this current significantly. DBEP (30 μM) had little or no effect on IK(erg), while DMGP (30 μM) slightly reduced it. In inside-out configuration, DEHP (30 μM) applied to the bath slightly reduced the activity of large-conductance Ca²⁺-activated K⁺ channels. DEHP (30 μM) increased the frequency of spontaneous action potentials (APs); however, this compound at the same concentration had no effect on AP firing in KCNH2 siRNA-transfected GH₃ cells. The effects described herein can contribute to their actions on functional activity of endocrine or neuroendocrine cells if similar results are found in vivo.

Ca2+ sensitivity of the maxi chloride channel in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) associated with the myenteric plexus of the small intestine express maxi chloride channels. Our aim was to investigate whether or not these channels would be activated by increases in intracellular Ca(2+) , as that would strengthen evidence for their potential role in ICC pacemaking. A further aim was to examine whether inwardly and outwardly rectifying maxi chloride currents signify different channels. We used Fluo-4 AM Ca(2+) imaging and patch clamp electrophysiology (cell-attached and inside-out) on isolated ICC in short term culture. Increasing intracellular Ca(2+) by three functionally distinct mechanisms (blocking sarcoplasmic reticulum Ca(2+) refilling, creating membrane Ca(2+) pores and a solution designed to block plasmalemmal Ca(2+) extrusion) was followed by inwardly rectifying maxi chloride channel activation assessed in the cell-attached configuration. Furthermore, in the inside-out configuration, increased outwardly rectifying maxi-chloride channel activity followed an increase in Ca(2+) to 2 mmol L(-1) at the cytoplasmic face of the channel. Increase in intracellular Ca(2+) will activate the maxi chloride channels. Maxi chloride currents are inwardly rectifying in the cell-attached patch clamp configuration under physiological conditions and are outwardly rectifying in the inside-out configuration. The same channel is responsible for both currents. Ca(2+) does not appear to regulate the rectification.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

Matrix Metalloproteinase-9 Increases the Ligand Sensitivity of CNG Channels: Evidence for Sequential Subunit Modification

Matrix metalloproteinases (MMPs) are a family of over 20 cell-surface and secreted, Zn2+- and Ca2+-dependent endopeptidases. Two closely related gelatinase MMPs, −9 and −2, have been implicated in both normal retinal processes and in several retinal diseases. MMPs are classically associated with tissue remodeling, but have been shown to influence ion channel function—either indirectly (e.g., through modification of the pericellular environment) or directly (e.g., by proteolysis of channel subunits). Because MMPs have been localized within the interphotoreceptor matrix, we hypothesized that MMP catalytic activity can influence the function of photoreceptor cyclic nucleotide-gated (CNG) channels. Cone and rod CNG channels were individually expressed in Xenopus oocytes and examined via patch-clamp recording in the inside-out configuration. Extracellular exposure to MMP-9 or −2 produced a profound increase in the efficacy of cAMP and the apparent cGMP affinity for both homomeric (CNGA3, K1/2,GMP, mean ± S.E.M: MMP960min = 0.62 μM ± 0.21; control60min = 12.6 μM ± 1.7) and heteromeric (CNGA3+CNGB3, K1/2,GMP: MMP980min = 2.2 μM ± 0.6; control80min = 16.4 μM ± 1.2) channel compositions. The gating changes required MMP-catalytic activity, exhibited complex state-dependence and were attenuated by N-glycosylation in the pore turret of CNGA subunits. Furthermore, MMP exposure resulted in decreased abundance of the full length CNGA3 subunit on immunoblots, and a concomitant increase in putative degradation products, consistent with proteolysis of core-channel subunits. Analyses of cGMP dose-response curves and current fluctuations suggest that MMPs generate channel subpopulations by sequentially modifying individual CNG channel subunits, where the apparent affinity for cGMP is governed by the total number of modified subunits (from 0 to 4). Together, these results provide evidence that MMPs can incrementally regulate the ligand sensitivity of CNG channels by proteolytic modification of CNGA subunits.

Differential Effects of Beta Subunits on the Voltage Sensor of Ca2+ and Voltage-Activated K+ Channels

Large conductance, Ca2+-, and voltage-activated K+ (BK) channels are tetramers composed by four pore-forming subunit (α) and four modulatory β-subunits. BK channels can be activated independently by membrane depolarization or an increase in cytosolic Ca2+. In particular, the presence of the β1-subunit modulates several aspect of channel activity, including the apparent Ca2+ sensitivity and the speed of activation and deactivation. Here, through the measurements of gating currents, we explored the effect of β-subunits on the voltage sensor of the BK channel using macropatches of Xenopus oocyte membrane in the inside-out configuration. Gating currents were recorded in solutions free of permeant ions and integrated during the onset of depolarizing pulses to generated charge-movement versus voltage (Q(V)) curves. Co-expression of the human BK α subunit with β1, resulted in about 70 mV left shift of the Q(V) curve, as compared to those obtained with the α-subunit alone or together with other β-subunits (2,3 & 4). To identify the molecular determinants that confer to the β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeras by exchanging the N- and C-terminus, transmembrane segments, and the extracellular loop between β1 and β2 subunits. Each of this construct alters the effects of the wild-type β1 subunit on the BK channel. Our results show that only β1 affects the workings of the BK voltage sensor and that the cytoplasmic N- and C-terminal and the external loop are necessary to recover the β1 subunit phenotype. On the basis of these results, we propose a possible molecular model where the whole β1 subunit is interacting with the voltage sensor of α-subunit in BK. Supported by Fondecyt grants 1110430 to R.L, 1980635 to AN. CINV is a Millennium Scientific Institute.

Decrease in phosphatidylinositol 4,5‐bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca(2+), a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] levels. The decrease in PtdIns(4,5)P(2) levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP(3)) production, and was inhibited by loading the cells with the Ca(2+) chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca(2+) concentrations. PtdIns(4,5)P(2) hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P(2) level measurements. Intracellular dialysis of PtdIns(4,5)P(2) inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P(2), did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P(2) using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca(2+) influx through TRPM8 activates a Ca(2+)-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P(2) plays a major role in desensitization of both cold and menthol responses.

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels

AimsDexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K+ (KATP) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular KATP-channel activity at the single-channel level. Main methodsWe used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular KATP channels, recombinant KATP channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels. Key findingsDexmedetomidine was observed to inhibit the native rat vascular KATP channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 KATP channels as well as Kir6.2ΔC36 channels with equivalent potency. SignificanceThese results indicate that dexmedetomidine directly inhibits KATP channels through the Kir6.0 subunit.

Molecular Mechanisms of the Inhibitory Effects of Clonidine on Vascular Adenosine Triphosphate–Sensitive Potassium Channels

We investigated the effects of the imidazoline-derived α2-adrenoceptor agonist clonidine on vascular adenosine triphosphate-sensitive potassium (K(ATP)) channel activity in rat vascular smooth muscle cells and recombinant vascular K(ATP) channels transiently expressed in COS-7 cells. Using the patch-clamp method, we investigated the effects of clonidine on the following: (1) native vascular K(ATP) channels; (2) recombinant K(ATP) channels with different combinations of various types of inwardly rectifying potassium channel (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor (SUR1, 2A, 2B) subunits; (3) SUR-deficient channels derived from a truncated isoform of the Kir6.2 subunit (Kir6.2ΔC36 channels); and (4) mutant Kir6.2ΔC36 channels with diminished sensitivity to ATP (Kir6.2ΔC36-K185Q channels). Clonidine (≥3 × 10(-8) M) inhibited native K(ATP) channel activity in cell-attached configurations with a half-maximal inhibitory concentration value of 1.21 × 10(-6) M and in inside-out configurations with a half-maximal inhibitory concentration value of 0.89 × 10(-6) M. With similar potency, clonidine (10(-6) or 10(-3) M) also inhibited the activities of various recombinant SUR/Kir6.0 K(ATP) channels, the Kir6.2ΔC36 channel, and the Kir6.2ΔC36-K185Q channel. Clinically relevant concentrations of clonidine inhibit K(ATP) channel activity in vascular smooth muscle cells. This inhibition seems to be the result of its effect on the Kir6.0 subunit and not on the SUR subunit.