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The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

We have previously shown that in rats, capsaicin (Cap) has antidepressant-like properties when assessed using the forced swimming test (FST) and that a sub-threshold dose of amitriptyline potentiates the effects of Cap. However, synergistic antidepressant-like effects of the joint administration of Cap and the selective serotonin reuptake inhibitor citalopram (Cit) have not been reported. To assess whether combined administration of Cap and Cit has synergistic effects in the FST and to determine whether this combination prevents the side effects of Cit. Cap, Cit, and the co-administration of both substances were evaluated in a modified version of the FST (30-cm water depth) conducted in rats, as well as in the open field test (OFT), elevated plus maze (EPM), and Morris water maze (MWM). In line with previous studies, independent administration of Cap and Cit displayed antidepressant-like properties in the FST, while the combined injection had synergistic effects. In the OFT, neither treatment caused significant increments in locomotion. In the EPM, the time spent in the closed arms was lower in groups administered either only Cap or a combination of Cap and Cit than in groups treated with Cit alone. In the MWM, both Cap and the joint treatment (Cap and Cit) improved the working memory of rats in comparison with animals treated only with Cit. Combined administration of Cap and Cit produces a synergistic antidepressant-like effect in the FST and reduces the detrimental effects of Cit on anxiety and working memory.

The suprachiasmatic nucleus (SCN) is the main brain clock in mammals. The SCN synchronizes to the light-dark cycle through the retinohypothalamic tract (RHT). RHT axons release glutamate to activate AMPA-kainate and N-methyl-D-aspartate (NMDA) postsynaptic receptors in ventral SCN neurons. Stimulation of SCN NMDA receptors is necessary for the activation of the signaling cascades that govern the advances and delays of phase. To our knowledge, no research has been performed to analyze the functional synaptic modifications occurring during postnatal development that prepare the circadian system for a proper synchronization to light at adult ages. Here, we studied the pre- and postsynaptic developmental changes between the unmyelinated RHT-SCN connections. Spontaneous NMDA excitatory postsynaptic currents (EPSCs) were greater in amplitude and frequency at postnatal day 34 (P34) than at P8. Similarly, both quantal EPSCs (miniature NMDA and evoked quantal AMPA-kainate) showed a development-dependent increase at analyzed stages, P3-5, P7-9, and P13-18. Moreover, the electrically evoked NMDA and AMPA-kainate components were augmented with age, although the increment was larger for the latter, and the membrane resting potential was more depolarized at early postnatal ages. Finally, the short-term synaptic plasticity was significantly modified during postnatal development as was the estimated number of quanta released and the initial release probability. All of these synaptic modifications in the unmyelinated RHT-SCN synapses suggest that synchronization to light at adult ages requires developmental changes similar to those that occur in myelinated fast communication systems.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

Terfenadine is a second-generation H1-antihistamine that despite potentially can produce severe side effects it has recently gained attention due to its anticancer properties. Lately, the subfamily 2 of inward rectifier potassium channels (Kir2) has been implicated in the progression of some tumoral processes. Hence, we characterized the effects of terfenadine on Kir2.x channels expressed in HEK-293 cells. Terfenadine inhibited Kir2.3 channels with a strikingly greater potency (IC 50 = 1.06 ± 0.11 μmol L−1) compared to Kir2.1 channels (IC 50 = 27.8 ± 4.8 μmol L−1). The Kir2.3(I213L) mutant, possessing a larger affinity for phosphatidylinositol 4,5-bisphosphate (PIP2) than the wild-type Kir2.3, was less sensitive to terfenadine inhibition (IC 50 = 13.0 ± 2.9 μmol L−1). Additionally, the PIP2 intracellular application had largely reduced the inhibition of Kir2.1 channels by terfenadine. Our data support that Kir2.x channels are targets of terfena-dine by affecting their interaction with PIP2, which could be regarded as a mechanism of the antitumor properties of terfenadine.

Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 μM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 μM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.

Transient receptor potential vanilloid 1 (TRPV1) channels have been implicated in depression and anxiety. The aim of this study was to evaluate the antidepressant-like properties of the TRPV1 agonist capsaicin using the forced swimming test (FST) in rats. Capsaicin (0.001–0.25 mg/kg, i.p.) produced a reduction of immobility in the FST. A maximally effective dose of the tricyclic antidepressant amitriptyline (12 mg/kg) reduced immobility as well. Notably, doses of capsaicin (1 pg/kg, 1 ng/kg, and 0.001 mg/kg) that were ineffective when applied alone produced a significant decrease in immobility when combined with a subthreshold dose of amitriptyline (5 mg/kg). Rats treated with capsaicin (0.01 mg/kg) + amitriptyline (5 mg/kg) displayed less immobility than those treated with a maximally effective dose of amitriptyline. The non-pungent TRPV1 channel agonist palvanil (0.05–0.1 mg/kg, i.p.) also decreased immobility in the FST. Capsaicin (0.05 mg/kg) did not affect general locomotion in the open field test, nor performance in the elevated plus maze, or skeletal muscle contraction strength measured in vitro after the FST (at 0.25 mg/kg). Altogether, our results imply that low doses of capsaicin produce antidepressant-like effects, and enhance the effect of a subthreshold dose of amitriptyline in the FST.

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Amitriptyline (AMIT) is a compound widely prescribed for psychiatric and non-psychiatric conditions including depression, migraine, chronic pain, and anorexia. However, AMIT has been associated with risks of cardiac arrhythmia and sudden death since it can induce prolongation of the QT interval on the surface electrocardiogram and torsade de pointes ventricular arrhythmia. These complications have been attributed to the inhibition of the rapid delayed rectifier potassium current (IKr). The slow delayed rectifier potassium current (IKs) is the main repolarizing cardiac current when IKr is compromised and it has an important role in cardiac repolarization at fast heart rates induced by an elevated sympathetic tone. Therefore, we sought to characterize the effects of AMIT on Kv7.1/KCNE1 and homomeric Kv7.1 channels expressed in HEK-293H cells. Homomeric Kv7.1 and Kv7.1/KCNE1 channels were inhibited by AMIT in a concentration-dependent manner with IC50 values of 8.8 ± 2.1 μM and 2.5 ± 0.8 μM, respectively. This effect was voltage-independent for both homomeric Kv7.1 and Kv7.1/KCNE1 channels. Moreover, mutation of residues located on the P-loop and S6 domain along with molecular docking, suggest that T312, I337 and F340 are the most important molecular determinants for AMIT-Kv7.1 channel interaction. Our experimental findings and modeling suggest that AMIT preferentially blocks the open state of Kv7.1/KCNE1 channels by interacting with specific residues that were previously reported to be important for binding of other compounds, such as chromanol 293B and the benzodiazepine L7.

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

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 (IC 50 ≈0.5 μM at +50 mV) and faster onset of block when compared to whole-cell configuration (IC 50 ≈7 μM 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.

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.

It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (IKACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated IKACh with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (τ = 14.8 ± 2.3 s) deactivation kinetics of the IXO-evoked IKACh than that at the positive voltage (τ = 6.7 ± 1.2 s). The voltage-dependent M2R-IXO interaction induced IKACh to exhibit voltage-dependent features of this current, such as the ‘relaxation gating’ and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the ‘tyrosine lid’ (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.

The suprachiasmatic nucleus (SCN) is the main brain clock that regulates circadian rhythms in mammals. The SCN synchronizes to the LD cycle through the retinohypothalamic tract (RHT), which projects to ventral SCN neurons via glutamatergic synapses. Released glutamate activates N-methyl-D-aspartate (NMDA) receptors, which play a critical role in the activation of signaling cascades to enable phase shifts. Previous evidence indicates that presynaptic changes during postnatal development consist of an increase in RHT fibers impinging on SCN neurons between postnatal day (P) 1 to 4 and P15. The aim of this study was to evaluate postsynaptic developmental changes in the NR2 subunits that determine the pharmacological and biophysical properties of the neuronal NMDA receptors in the ventral SCN. To identify the expression of NR2 subtypes, we utilized RT-PCR, immunohistochemical fluorescence, and electrophysiological recordings of synaptic activity. We identified development-dependent changes in NR2A, C, and D subtypes in mRNA and protein expression, whereas NR2B protein was equally present at all analyzed postnatal ages. The NR2A antagonist PEAQX (100 nM) reduced the frequency of NMDA excitatory postsynaptic currents (EPSCs) at P8 significantly more than at P34, but the antagonists for NR2B (3 μM Ro 25-6981) and NR2C/D (150 nM PPDA) did not influence NMDA EPSCs differently at the 2 analyzed postnatal ages. Our results point to P8 as the earliest analyzed postnatal age that shows mRNA and protein expression similar to those found at the juvenile stage P34. Taken together, our findings indicate that postsynaptic development-dependent modifications in the NR2 subtypes of the NMDA receptor could be important for the synchronization of ventral SCN neurons to the LD cycle at adult stages.

The cardiac muscarinic receptor (M2R) regulates heart rate, in part, by modulating the acetylcholine (ACh) activated K+ current IK,ACh through dissociation of G-proteins, that in turn activate KACh channels. Recently, M2Rs were noted to exhibit intrinsic voltage sensitivity, i.e. their affinity for ligands varies in a voltage dependent manner. The voltage sensitivity of M2R implies that the affinity for ACh (and thus the ACh effect) varies throughout the time course of a cardiac electrical cycle. The aim of this study was to investigate the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potentials in physiological and pathophysiological conditions. We developed a Markovian model of the IK,ACh modulation by voltage and integrated it into a computational model of human sinus node. We performed simulations with the integrated model varying ACh concentration and voltage sensitivity. Low ACh exerted a larger effect on IK,ACh at hyperpolarized versus depolarized membrane voltages. This led to a slowing of the pacemaker rate due to an attenuated slope of phase 4 depolarization with only marginal effect on action potential duration and amplitude. We also simulated the theoretical effects of genetic variants that alter the voltage sensitivity of M2R. Modest negative shifts in voltage sensitivity, predicted to increase the affinity of the receptor for ACh, slowed the rate of phase 4 depolarization and slowed heart rate, while modest positive shifts increased heart rate. These simulations support our hypothesis that altered M2R voltage sensitivity contributes to disease and provide a novel mechanistic foundation to study clinical disorders such as atrial fibrillation and inappropriate sinus tachycardia., Author summary Heart rate regulation is dependent upon a delicate interplay between parasympathetic and sympathetic nerve activity at the level of the sinus node. Acetylcholine slows the heart rate by activating the M2 muscarinic receptor (M2R) that, in turn, opens the acetylcholine-activated potassium channel (IK,ACh) to slow the firing of the sinus node. Surprisingly, the M2R is sensitive to membrane potential and undergoes conformational changes throughout the cardiac action potential that alter the affinity for acetylcholine, with secondary consequences for IK,ACh activity. Here, we investigated the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potential in physiological and pathophysiological conditions, using a Markovian model of the IK,ACh channel integrated into a computational model of human sinus node. The computational model allowed us to assess the effects of potential genetic variants that alter specific properties of voltage sensitivity. Our results indicate that alterations in the M2R voltage sensitivity play a significant role in the physiology and pathophysiology of the human sinus node and atria. Our computational model is relevant for future studies aimed at the design and development of anti-arrhythmic agents that specifically target the unique voltage-sensitive properties of M2R.

The acetylcholine (ACh)-gated inwardly rectifying K

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 ± 0.3 μM 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.

The human intestinal pathogen Giardia lamblia is a flagellated unicellular protozoan with pronounced medical and biological relevance. However, the basic physiology of Giardia trophozoites has been sparsely studied, especially the electrical and ionic properties of their cellular membrane which are virtually unknown. In this study, we were able to record and characterize the macroscopic ionic currents of Giardia trophozoite membrane by electrophysiological methods of the patch clamp technique. Giardia trophozoites showed a high current density (∼600 pA/pF at -140 mV) that was activated upon hyperpolarization. This current was carried by a chloride-selective channel (I Cl-G) and it was the most important determinant of the membrane potential in Giardia trophozoites. Moreover, this conductance was able to carry other halide anions and the sequence of permeability was Br(-) > Cl(-) ≈ I(-) ≫ F(-). Besides the voltage-dependent inward-rectifying nature of I Cl-G, its activation and deactivation kinetics were comparable to those observed in ClC-2 channels. Extracellular pH modified the voltage-dependent properties of I Cl-G, shifting the activation curve from a V 1/2 = -79 ± 1 mV (pH 7.4) to -93 ± 2 mV (pH 8.4) and -112 ± 2 mV (pH 5.4). Furthermore, the maximal amplitude of I Cl-G measured at -100 mV showed dependence to external pH in a bell-shaped fashion reported only for ClC-2 channels. Therefore, our results suggest that I Cl-G possesses several functional properties similar to the mammalian ClC-2 channels.

Choline(Ch)isaprecursorandmetaboliteoftheneurotransmitteracetylcholine(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 (IKACh )a nd showedthat,incontrasttoACh,pilocarpineinducesacurrentwithdelayedrectifier-likeproperties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependentmanner similar to pilocarpine. Single-channel recordings, biophysicalprofiles,specificpharmacologicalinhibitionandcomputationaldataindicatethatthe current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases 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.

Potassium (K(+)) channels are crucial for determining the shape, duration, and frequency of action-potential firing in excitable cells. Broadly speaking, K(+) channels can be classified based on whether their macroscopic current outwardly or inwardly rectifies, whereby rectification refers to a change in conductance with voltage. Outwardly rectifying K(+) channels conduct greater current at depolarized membrane potentials, whereas inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward currents through inwardly rectifying K(+) channels are reduced at more depolarized potentials. However, the acetylcholine-gated K(+) channel (KACh) conducts current that inwardly rectifies when activated by some ligands (such as acetylcholine), and yet conducts current that outwardly rectifies when activated by other ligands (for example, pilocarpine and choline). The perplexing and paradoxical behavior of KACh channels is due to the intrinsic voltage sensitivity of the receptor that activates KACh channels, the M2 muscarinic receptor (M2R). Emerging evidence reveals that the affinity of M2R for distinct ligands varies in a voltage-dependent and ligand-specific manner. These intrinsic receptor properties determine whether current conducted by KACh channels inwardly or outwardly rectifies. This review summarizes the most recent concepts regarding the intrinsic voltage sensitivity of muscarinic receptors and the consequences of this intriguing behavior on cardiac physiology and pharmacology of KACh 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, I(K1) and I(KATP), 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 I(KATP) I(K1). 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, PIP(2)-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 PIP(2) 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 PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) 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.

Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M(2) receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K(+) current (I (KACh)) 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 I (KACh). 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, I (KACh) in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of I (KACh).

The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, I(KACh). Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I(KACh) modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.

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 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 (IC(50) approximately 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial beta-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.

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 (PIP2). We tested the hypothesis that Kir channels with low affinity for PIP2, like the adenosine triphosphate (ATP)-sensitive K+ channel (KATP) and acetylcholine (ACh)-activated K+ channel (KACh), 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 (KATP) and Kir3.1/3.4 (KACh) channels expressed in HEK-293 cells and ATP-sensitive K+ current (IKATP) and ACh-activated K+ current (IKACh) in feline atrial myocytes. The onset of tamoxifen inhibition of both IKATP and IKACh was slow (T1/2 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 PIP2. In atrial myocytes, the mechanism and potency of the effects of tamoxifen on KATP and KACh channels were comparable to those in HEK-293 cells. These data suggest that, similar to its effects on Kir2.x currents, tamoxifen inhibits KATP and KACh currents by interfering with the interaction between the channel and PIP2. Keywords:: adenosine triphosphate–sensitive K+ current (IKATP), inhibition, K+ current activated by muscarinic agonist (IKACh), phosphatidylinositol 4,5-bisphosphate (PIP2), tamoxifen

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.

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 ~50 min. 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.

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.

The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, I(KACh). Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I(KACh) modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.

In addition to external stimuli, membrane voltage modulates the activity of several G-protein-coupled receptors (GPCRs). For example, membrane depolarization reduces the affinity of the M2 muscarinic receptor (M2R) for acetylcholine (ACh). The intrinsic capacity of muscarinic receptors to “sense” transmembrane potential was confirmed by the recording of “gating” currents that reflect reorientation of charges within the receptor in response to changes in voltage. We studied the effects of membrane voltage on agonist activation of M2R in atrial myocytes and on gating currents in oocytes heterologously expressing M2R. Depolarization decreased the apparent affinity of M2R for ACh, but increased the apparent affinity and efficacy of pilocarpine (PILO), as measured by acetylcholine-activated K+ current, IKACh. Voltage-induced conformational changes in M2R (as measured by gating currents) were modified in a ligand-selective manner. For example, ACh reduced gating charge displacement while PILO increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent IKACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor and confirmed a link between voltage sensing and changes in agonist affinity. Our data suggest that voltage modulates the conformation of the ligand binding site, while ligand binding induces ligand-specific conformational changes in the receptor. Voltage-dependent GPCR modulation has important implications for cellular signaling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany ligand binding and changes in membrane voltage.

Kv1.5 is considered to be a potential molecular target for treatment of atrial fibrillation or flutter. Disopyramide is widely used in the treatment of atrial flutter and/or atrial fibrillation. The present study was undertaken to characterize the effects of disopyramide on currents mediated by Kv1.5 channels and to determine the putative binding site involved in the inhibitory effects of disopyramide. Experiments were carried out on wild-type and site directed mutated hKv1.5 channels expressed on HEK 293 cells using the patch-clamp technique. Disopyramide acting from the cytoplasmic side of the membrane produced blocking effects on Kv1.5 that exhibited several features typical of an open channel blocker. Ala-scanning mutagenesis of the Kv1.5 pore domain combined with macroscopic current analysis suggested that disopyramide interacted only with the Val512 residue that faces to the central cavity of the channel. Mutation of this key residue to Ala caused marked change in the IC50 of disopyramide (22-fold). The single interaction between disopyramide and Val512 in the PVP region is able to change the mechanism of channel closure, reproducing the “foot-in-the-door” phenomenon. Keywords:: potassium channel, ultra rapid delayed rectifier, Kv1.5, antiarrhythmic, disopyramide

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 ~ 50 mm 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 (1 mM) 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.