Iván A. Aréchiga-Figueroa

Tamoxifen inhibits inward rectifier K+ 2.x family of inward rectifier channels by interfering with phosphatidylinositol 4,5-bisphosphate-channel interactions.

Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits the inward rectifier potassium current (IK1) in cardiac myocytes by an unknown mechanism. We characterized the inhibitory effects of tamoxifen on Kir2.1, Kir2.2, and Kir2.3 potassium channels that underlie cardiac IK1. We also studied the effects of 4-hydroxytamoxifen and raloxifene. All three drugs inhibited inward rectifier K+ 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 > Kir2.1 ∼ Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T1/2 ∼ 6 min) and only partially recovered after washout (∼30%). Kir2.x inhibition was concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. We tested the hypothesis that tamoxifen interferes with the interaction between the channel and the membrane-delimited channel activator, phosphatidylinositol 4,5-bisphosphate (PIP2). Inhibition of Kir2.3 currents was significantly reduced by a single point mutation of I213L, which enhances Kir2.3 interaction with membrane PIP2. Pretreatment with PIP2 significantly decreased the inhibition induced by tamoxifen, 4-hydroxytamoxifen, and raloxifene on Kir2.3 channels. Pretreatment with spermine (100 μM) decreased the inhibitory effect of tamoxifen on Kir2.1, probably by strengthening the channels interaction with PIP2. In cat atrial and ventricular myocytes, 3 μM tamoxifen inhibited IK1, but the effect was greater in the former than the latter. The data strongly suggest that tamoxifen, its metabolite, and the estrogen receptor inhibitor raloxifene inhibit Kir2.x channels indirectly by interfering with the interaction between the channel and PIP2.

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.

Carvedilol inhibits Kir2.3 channels by interference with PIP2-channel interaction

Carvedilol, a β- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. It has been proposed to block K(ATP) channels by binding to the bundle crossing region at a domain including cysteine at position 166, and thereby plugging the pore region. However, carvedilol was reported not to affect Kir2.1 channels, which lack 166 Cys. Here, we demonstrate that carvedilol inhibits Kir2.3 carried current by an alternative mechanism. Carvedilol inhibited Kir2.3 channels with at least 100 fold higher potency (IC(50)=0.49 μM) compared to that for Kir2.1 (IC(50)>50 μM). Kir2.3 channel inhibition was concentration-dependent and voltage-independent. Increasing Kir2.3 channel affinity for PIP(2), by a I213L point mutation, decreased the inhibitory effect of carvedilol more than twentyfold (IC(50)=11.1 μM). In the presence of exogenous PIP(2), Kir2.3 channel inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition). These results suggest that carvedilol, as other cationic amphiphilic drugs, inhibits Kir2.3 channels by interfering with the PIP(2)-channel interaction.

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.

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.

Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes

Background: The rapid delayed rectifier K+ current (IKr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Modulation of Kv2.1 channels inactivation by curcumin.

BACKGROUND The aim of the present study was to assess the effects of curcumin on the voltage-dependent Kv2.1 potassium channel. METHODS The whole-cell patch-clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by curcumin. RESULTS Curcumin reduced the Kv2.1 currents; the inhibition occurred with a slow time course and was partially reversible. Curcumin did not alter the kinetics and voltage dependence of activation; however, the kinetics of open- and closed-state inactivation was accelerated by curcumin along with a hyperpolarizing shift in the voltage dependence of inactivation. Curcumin inhibition of Kv2.1 current was not use-dependent. CONCLUSIONS Overall, our data suggest that curcumin inhibits Kv2.1 channels by modulating the inactivation gating, which would be expected to impact cellular physiology.

Tamoxifen Inhibition of Kv7.2/Kv7.3 Channels

KCNQ genes encode five Kv7 K+ channel subunits (Kv7.1–Kv7.5). Four of these (Kv7.2–Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.