Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits the inward rectifier potassium current (I K1 ) 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 I K1 . We also studied the effects of 4-hydroxytamoxifen and raloxifene. All three drugs inhibited inward rectifier K ϩ 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 Ͼ Kir2.1 ϳ Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T 1/2 ϳ 6 min) and only partially recovered after washout (ϳ30%). 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 (PIP 2 ). Inhibition of Kir2.3 currents was significantly reduced by a single point mutation of I213L, which enhances Kir2.3 interaction with membrane PIP 2 . Pretreatment with PIP 2 significantly decreased the inhibition induced by tamoxifen, 4-hydroxytamoxifen, and raloxifene on Kir2.3 channels. Pretreatment with spermine (100 M) decreased the inhibitory effect of tamoxifen on Kir2.1, probably by strengthening the channel's interaction with PIP 2 . In cat atrial and ventricular myocytes, 3 M tamoxifen inhibited I K1 , 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 PIP 2 .

Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca -dependent Cl channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H ] ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H ] from 10 to 10 m caused a progressive increase in the chloride current (I ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca ] . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the I was nearly insensitive to proton concentrations between 10 and 10 m. Additionally, I of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10 m, but 10 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca ] .

4-aminopyridine (4-AP) is commonly used to block the transient outward potassium current, I(to), in cardiac and noncardiac tissues. In the present work, we found that 4-AP inhibited the rapid component of the delayed rectifier potassium current, I(Kr), in rabbit-isolated sinoatrial node myocytes by 25% (1 mM) and 51% (5 mM) and inhibited the slow component of the delayed rectifier potassium current, I(Ks), in cat- isolated sinoatrial node myocytes by 39% (1 mM) and 62% (5 mM). In cat- and rabbit-isolated sinoatrial node myocytes, 4-AP activated muscarinic receptors in a voltage-dependent manner to increase the acetylcholine-activated potassium current, I(KACh). In multicellular preparations of the central region of the sinoatrial node from nonreserpinized rabbits, 4-AP produced an increase in action potential overshoot, frequency, and rate of diastolic depolarization. In the presence of the beta-adrenergic antagonist propranolol, 4-AP produced a marked increase in duration and a marked decrease in maximum diastolic potential and eventually, cessation of the spontaneous activity in preparations from the sinoatrial central region. In multicellular preparations from reserpinized rabbits, 4-AP produced similar effects to those observed in the presence of propranolol. We conclude that 4-AP inhibits multiple cardiac K(+) currents, including I(to), I(Kr), and I(Ks), and that these activities mask I(KACh) activation. In addition, in multicellular preparations, 4-AP produces neurotransmitter release from the autonomic nerve terminals. These multiple effects need to be considered when using 4-AP as a "specific" I(to) blocker.

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Key points• 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 (I KACh ).• Membrane depolarization increased the potency and efficacy of I KACh 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 I KACh modulation is both voltage-and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.Abstract Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K + current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand-and voltage-dependent properties of muscarinic activation of ACh-activated K + current (I KACh ) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates I KACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is I KACh . Moreover, we show that membrane depolarization increases the potency and efficacy of I KACh 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 I KACh modulation is both voltage-and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.