Eloy G. Moreno-Galindo

Relaxation gating of the acetylcholine-activated inward rectifier K + current is mediated by intrinsic voltage sensitivity of the muscarinic receptor

Non‐technical summary  Normal heart rate variability is critically dependent upon the G‐protein‐coupled, acetylcholine (ACh)‐activated inward rectifier K+ current, IKACh. A unique feature of IKACh is the so‐called ‘relaxation’ gating property that contributes to increased current at hyperpolarized membrane potentials. Here, we consider a novel explanation for IKACh relaxation based upon the recent finding that G‐protein‐coupled receptors are intrinsically voltage sensitive and that the muscarinic agonists acetylcholine and pilocarpine manifest opposite voltage‐dependent IKACh modulation. Based on experimental and computational findings, we propose that IKACh relaxation represents a voltage‐dependent change in agonist affinity as a consequence of a voltage‐dependent conformational change in the muscarinic receptor.

The antimalarial drug mefloquine inhibits cardiac inward rectifier K+ channels: evidence for interference in PIP2-channel interaction.

The antimalarial drug mefloquine was found to inhibit the KATP channel by an unknown mechanism. Because mefloquine is a Cationic amphiphilic drug and is known to insert into lipid bilayers, we postulate that mefloquine interferes with the interaction between PIP2 and Kir channels resulting in channel inhibition. We studied the inhibitory effects of mefloquine on Kir2.1, Kir2.3, Kir2.3(I213L), and Kir6.2/SUR2A channels expressed in HEK-293 cells, and on IK1 and IKATP from feline cardiac myocytes. The order of mefloquine inhibition was Kir6.2/SUR2A ≈ Kir2.3 (IC50 ≈ 2 μM) > Kir2.1 (IC50 > 30 μM). Similar results were obtained in cardiac myocytes. The Kir2.3(I213L) mutant, which enhances the strength of interaction with PIP2 (compared to WT), was significantly less sensitive (IC50 = 9 μM). In inside-out patches, continuous application of PIP2 strikingly prevented the mefloquine inhibition. Our results support the idea that mefloquine interferes with PIP2-Kir channels interactions.

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, IK1 and IKATP, in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited IKATP > IK1. We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP2–Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP2 is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP2 and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP2 increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP2 lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.

Mechanosensitive Ca2 +-permeable channels in human leukemic cells: Pharmacological and molecular evidence for TRPV2

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²⁺ and Mg²⁺. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³⁺ (10 μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 μM) and SKF 96365 (250 μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²⁺ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

Molecular Basis for a High-Potency Open-Channel Block of Kv1.5 Channel by the Endocannabinoid Anandamide

The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K+ (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC50 ≈ 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial β-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K+ channel families.

Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier-like activation of ACh-gated K(+) current by choline in feline atrial myocytes.

•  Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). •  Previously, in cardiomyocytes Ch was shown to activate an outward K+ current in a delayed rectifier fashion, which has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the identity of this current remains elusive. •  Single‐channel recordings, biophysical profiles and specific pharmacological inhibition indicate that the current activated by Ch is the ACh‐activated K+ current (IKACh). •  Membrane depolarization increased the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K+ current at depolarized potentials. •  Our findings support the emerging concept that IKACh modulation is both voltage‐ and ligand‐specific and reinforce the importance of these properties in understanding cardiac physiology.

Muscarinic-activated potassium current mediates the negative chronotropic effect of pilocarpine on the rabbit sinoatrial node

Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M2 receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K+ current (IKACh) on the negative chronotropic effect of pilocarpine in rabbit sinoatrial node. In multicellular preparations, we studied the effect of pilocarpine on spontaneous action potentials. In isolated myocytes, using the patch clamp technique, we studied the effects of pilocarpine on IKACh. Pilocarpine produced a decrease in spontaneous frequency, hyperpolarization of the maximum diastolic potential, and a decrease in the diastolic depolarization rate. These effects were partially antagonized by tertiapin Q. Cesium and calyculin A in the presence of tertiapin Q partially prevented the effects of pilocarpine. In isolated myocytes, pilocarpine activated the muscarinic potassium current, IKACh in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of IKACh.

Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism

ABSTRACT Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch‐clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK‐293 cells. CQ produced a fast‐onset voltage‐dependent pore‐blocking effect on these channels. In inside‐out patches, CQ showed notable higher potency (IC50 ≈0.5 &mgr;M at +50 mV) and faster onset of block when compared to whole‐cell configuration (IC50 ≈7 &mgr;M at +60 mV). Also, CQ showed a voltage‐dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore‐plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ‐Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.

Impact of the whole-cell patch-clamp configuration on the pharmacological assessment of the hERG channel: trazodone as a case example.

INTRODUCTION Voltage- and state-dependent blocks are important mechanisms by which drugs affect voltage-gated ionic channels. However, spontaneous (i.e. drug-free) time-dependent changes in the activation and inactivation of hERG and Na(+) channels have been reported when using conventional whole-cell patch-clamp in HEK-293 cells. METHODS hERG channels were heterologously expressed in HEK-293 cells and in Xenopus laevis oocytes. hERG current (IhERG) was recorded using both conventional and perforated whole-cell patch-clamp (HEK-293 cells), and two microelectrode voltage-clamp (Xenopus oocytes) in drug-free solution, and in the presence of the drug trazodone. RESULTS In conventional whole-cell setup, we observed a spontaneous time-dependent hyperpolarizing shift in the activation curve of IhERG. Conversely, in perforated patch whole-cell (HEK-293 cells) or in two microelectrode voltage-clamp (Xenopus oocytes) activation curves of IhERG were very stable for periods ~50min. Voltage-dependent inactivation of IhERG was not significantly altered in the three voltage clamp configurations tested. When comparing voltage- and state-dependent effects of the antidepressant drug trazodone on IhERG, similar changes between the three voltage clamp configurations were observed as under drug-free conditions. DISCUSSION The comparative analysis performed in this work showed that only under conventional whole-cell voltage-clamp conditions, a leftward shift in the activation curve of IhERG occurred, both in the presence and absence of drugs. These spontaneous time-dependent changes in the voltage activation gate of IhERG are a potential confounder in pharmacological studies on hERG channels expressed in HEK-293 cells.

Inhibition of Kir4.1 potassium channels by quinacrine

Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3μM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.