Non-technical summary Normal heart rate variability is critically dependent upon the G-protein-coupled, acetylcholine (ACh)-activated inward rectifier K + current, I KACh . A unique feature of I KACh is the so-called 'relaxation' gating property that contributes to increased current at hyperpolarized membrane potentials. Here, we consider a novel explanation for I KACh 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 I KACh modulation. Based on experimental and computational findings, we propose that I KACh relaxation represents a voltage-dependent change in agonist affinity as a consequence of a voltage-dependent conformational change in the muscarinic receptor.Abstract Normal heart rate variability is critically dependent upon the G-protein-coupled, acetylcholine (ACh)-activated inward rectifier K + current, I KACh . A unique feature of I KACh is the so-called 'relaxation' gating property that contributes to increased current at hyperpolarized membrane potentials. I KACh relaxation refers to a slow decrease or increase in current magnitude with depolarization or hyperpolarization, respectively. The molecular mechanism underlying this perplexing gating behaviour remains unclear. Here, we consider a novel explanation for I KACh relaxation based upon the recent finding that G-protein-coupled receptors (GPCRs) are intrinsically voltage sensitive and that the muscarinic agonists acetylcholine (ACh) and pilocarpine (Pilo) manifest opposite voltage-dependent I KACh modulation. We show that Pilo activation of I KACh displays relaxation characteristics opposite to that of ACh. We explain the opposite effects of ACh and Pilo using Markov models of I KACh that incorporate ligand-specific, voltage-dependent parameters. Based on experimental and computational findings, we propose a novel molecular mechanism to describe the enigmatic relaxation gating process: I KACh relaxation represents a voltage-dependent change in agonist affinity as a consequence of a voltage-dependent conformational change in the muscarinic receptor.

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.

Abstract. Tamoxifen inhibits transmembrane currents of the Kir2.x inward rectifier potassium channels by interfering with the interaction of the channels with membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We tested the hypothesis that Kir channels with low affinity for PIP 2 , like the adenosine triphosphate (ATP)-sensitive K + channel (K ATP ) and acetylcholine (ACh)-activated K + channel (K ACh ), have at least the same sensitivity to tamoxifen as Kir2.3. We investigated the effects of tamoxifen (0.1 -10 μM) on Kir6.2/SUR2A (K ATP ) and Kir3.1/3.4 (K ACh ) channels expressed in HEK-293 cells and ATP-sensitive K + current (I KATP ) and ACh-activated K + current (I KACh ) in feline atrial myocytes. The onset of tamoxifen inhibition of both I KATP and I KACh was slow (T 1/2 approximately 3.5 min) and concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. Tamoxifen interacts with the pore forming subunit, Kir6.2, rather than with the SUR subunit. The inhibitory potency of tamoxifen on the Kir6.2/SUR2A channel was decreased by the mutation (C166S) on Kir6.2 and in the continuous presence of PIP 2 . In atrial myocytes, the mechanism and potency of the effects of tamoxifen on K ATP and K ACh channels were comparable to those in HEK-293 cells. These data suggest that, similar to its effects on Kir2.x currents, tamoxifen inhibits K ATP and K ACh currents by interfering with the interaction between the channel and PIP 2 .

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.

Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K(+) current (I KACh) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I KACh by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I KACh was voltage insensitive. By contrast, deactivation of the ACh-induced I KACh was significantly slower at -100 mV than at +50 mV, while an opposite effect was observed when I KACh was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs.