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Background and purpose: Resting superior cervical ganglion (SCG) neurones are phasic cells that switch to a tonic mode of firing upon muscarinic receptor stimulation. This effect is partially due to the muscarinic inhibition of the M‐current. Because delayed rectifier K+ channels are essential to sustain tonic firing in central neurones, we asked whether the delayed rectifier current IKV in SCG neurones was modulated by the muscarinic receptors expressed in these cells. Experimental approach: Whole‐cell patch‐clamp records of M‐current and IKV were done in cultured or acutely dissociated rat SCG neurones. To characterize the receptor that regulates IKV, cells were bathed with muscarinic agonists and antagonists, relatively specific for receptor subtypes. Key results: The muscarinic agonist oxotremorine‐M (Oxo‐M) enhanced IKV by ∼46% relative to its basal value. This effect remained unaltered when M‐current was suppressed by linopirdine or Ba2+. Enhancement of IKV was insensitive to the M1‐antagonist pirenzepine, whereas it was inhibited (∼60%) by the M2/4‐antagonist himbacine. Further, the relatively specific M2‐agonist bethanechol was as potent as Oxo‐M in enhancing IKV. The modulation of IKV was insensitive to pertussis toxin (PTX), but was severely attenuated when internal ATP was replaced by its non‐hydrolysable analogue AMP‐PNP. Conclusions and Implications: These results suggest that an M2‐like muscarinic receptor couples to a PTX‐insensitive G‐protein and to an ATP‐dependent pathway to enhance IKV. Modulation of IKV must be taken into consideration in order to understand more precisely how muscarinic receptors acting on different ion channels regulate sympathetic excitability. British Journal of Pharmacology (2006) 149, 441–449. doi:10.1038/sj.bjp.0706874

Sympathetic activation robustly increases the slow delayed rectifier K + current (I Ks ) in the mammalian ventricular myocardium, however, exact downstream pathways involved in the β-adrenergic regulation of the current are not fully elucidated yet. This study examined the Ca 2+ sensitivity of I Ks and the contribution of the protein kinase A (PKA) and the calcium/calmodulin kinase II (CaMKII) pathways in regulating I Ks in isolated canine ventricular myocytes. Experiments were carried out under ß-adrenergic receptor activation (10 nM isoproterenol) and under baseline conditions (without isoproterenol). I Ks was measured as an HMR-1556 sensitive current with the action potential voltage clamp technique under the physiological intracellular calcium homeostasis of the cells. Reducing intracellular Ca 2+ concentration ([Ca 2+ ] i ) with 1 µM nisoldipine decreased the peak and mid-plateau densities of I Ks , reduced the current integral, and increased the time-to-peak value. In contrast, ß-adrenergic receptor activation by isoproterenol resulted in larger I Ks densities and integral, and shorter time-to-peak value. These effects of isoproterenol on I Ks were significantly smaller when the CaMKII inhibitor 1 µM KN-93 was present in the cells, but the PKA inhibitor 3 µM H-89 did not exert such effect. Importantly, all effects of isoproterenol on I Ks have fully developed even in the presence of 1 µM nisoldipine. Under baseline conditions the mid-plateau density of I Ks , was significantly smaller in the presence of KN-93, H-89 or nisoldipine, while peak I Ks density and the current integral were significantly smaller only in nisoldipine. In conclusion, many different signaling pathways are involved in regulating I Ks . Under baseline conditions the regulation is strongly [Ca 2+ ] i -dependent, with PKA and CaM-CaMKII involved, whereas during ß-adrenergic stimulation it is [Ca 2+ ] i -independent and supposes a pivotal role of EPAC-mediated activation of CaMKII.

Background: Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. Methods: Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH 3 ) cells were evaluated by patch clamp techniques. Results: ART inhibited the amplitude of delayed-rectifier K + current (I K(DR) ) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on I K(DR) with an IC 50 value of 11.2 µM and enhanced I K(DR) inactivation with a K D value of 14.7 µM. The steady-state inactivation curve of I K(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of I K(DR) in GH 3 cells. ART also decreased the peak amplitude of voltage-gated Na + current (I Na ) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late I Na , was effective at reversing ART-induced prolongation in inactivation time constant of I Na . Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. Conclusion: Under ART exposure, the inhibitory actions on both I K(DR) and I Na could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo.

Remdesivir (RDV, GS-5734), a broad-spectrum antiviral drug in the class of nucleotide analogs, has been particularly tailored for treatment of coronavirus infections. However, to which extent RDV is able to modify various types of membrane ion currents remains largely uncertain. In this study, we hence intended to explore the possible perturbations of RDV on ionic currents endogenous in pituitary GH cells and Jurkat T-lymphocytes. The whole-cell current recordings of ours disclosed that upon membrane depolarization in GH cells the exposure to RDV concentration-dependently depressed the peak or late components of elicitation with effective IC values of 10.1 or 2.8 μM, respectively; meanwhile, the value of dissociation constant of RDV-induced blockage of on the basis of the first-order reaction was yielded to be 3.04 μM. Upon the existence of RDV, the steady-state inactivation curve of was established in the RDV presence; moreover, the recovery became slowed. However, RDV-induced blockage of failed to be overcome by further addition of either α,β-methylene ATP or cyclopentyl-1,3-dipropylxanthine. The RDV addition also lessened the strength of M-type K current with the IC value of 2.5 μM. The magnitude of voltage hysteresis of elicited by long-lasting triangular ramp pulse was diminished by adding RDV. Membrane electroporation-induced current in response to large hyperpolarization was enhanced, with an EC value of 5.8 μM. Likewise, in Jurkat T-lymphocytes, adding RDV declined amplitude concomitantly with the raised rate of current inactivation applied by step depolarization. Therefore, in terms of the RDV molecule, there appears to be an unintended activity of the prodrug on ion channels. Its inhibition of both and occurring in a non-genomic fashion might provide additional but important mechanisms through which cellular functions are seriously perturbed.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0–+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used.

Despite advancement in anti‐seizure medications, 30% of patients continue to experience recurrent seizures. Previous data indicated the antiepileptic properties of melatonin and its agonists in several animal models. However, the underlying mechanisms of melatonin and its agonists on cellular excitability remain poorly understood. In this study, we demonstrated the electrophysiological changes of two main kinds of ion channels that are responsible for hyperexcitability of neurons after introduction of melatonin agonists‐ ramelteon (RAM). In Neuro‐2a cells, the amplitude of voltage‐gated Na+ (INa) and delayed‐rectifier K+ currents (IK (DR)) could be suppressed under RAM. The IC50 values of 8.7 and 2.9 μM, respectively. RAM also diminished the magnitude of window Na+ current (INa (W)) elicited by short ascending ramp voltage, with unchanged the overall steady‐state current–voltage relationship. The decaying time course of INa during a train of depolarizing pulses arose upon the exposure to RAM. The conditioning train protocol which blocked INa fitted the recovery time course into two exponential processes and increased the fast and slow time constant of recovery the presence of RAM. In pituitary tumor (GH3) cells, INa amplitude was also effectively suppressed by the RAM. In addition, GH3‐cells exposure to RAM decreased the firing frequency of spontaneous action potentials observed under current‐clamp conditions. As a result, the RAM‐mediated effect on INa was closely associated with its ability to decrease spontaneous action potentials. Collectively, we found the direct attenuation of INa and IK (DR) caused by RAM besides the agonistic action on melatonin receptors, which could partially explain its anti‐seizure activity. Ramelteon, a melatonin agonist, could attenuate sodium currents under whole‐cell patch clamp recording in neural cells. The changes in plasmalemmal ionic currents may influence the functional activities of different excitable cells and warrant further investigations.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Cortical and hippocampal hyperexcitability intervenes in the pathological derangement of brain activity leading to cognitive decline. As key regulators of neuronal excitability, the voltage-gated K+ channels (KV) might play a crucial role in the AD pathophysiology. Among them, the KV2.1 channel, the main α subunit mediating the delayed rectifier K+ currents (IDR) and controlling the intrinsic excitability of pyramidal neurons, has been poorly examined in AD. In the present study, we investigated the KV2.1 protein expression and activity in hippocampal neurons from the Tg2576 mouse, a widely used transgenic model of AD. To this aim we performed whole-cell patch-clamp recordings, Western blotting, and immunofluorescence analyses. Our Western blotting results reveal that KV2.1 was overexpressed in the hippocampus of 3-month-old Tg2576 mice and in primary hippocampal neurons from Tg2576 mouse embryos compared with the WT counterparts. Electrophysiological experiments unveiled that the whole IDR were reduced in the Tg2576 primary neurons compared with the WT neurons, and that this reduction was due to the loss of the KV2.1 current component. Moreover, we found that the reduction of the KV2.1-mediated currents was due to increased channel clustering, and that glutamate, a stimulus inducing KV2.1 declustering, was able to restore the IDR to levels comparable to those of the WT neurons. These findings add new information about the dysregulation of ionic homeostasis in the Tg2576 AD mouse model and identify KV2.1 as a possible player in the AD-related alterations of neuronal excitability.

Vortioxetine (VOR) is recognized to exert antidepressant actions. However, whether this drug modifies ionic currents in excitable cells remains unclear. The aim of this study was to explore the electrophysiological effects of VOR and other related compounds in pituitary GH3 cells and in Neuro-2a cells. VOR suppressed the delayed-rectifier K+ current (IK(DR)) in a concentration-, time-, and state-dependent manner. Effective IC50 values needed to inhibit peak and sustained IK(DR) were computed to be 31.2 and 8.5 μM, respectively, while the KD value estimated from minimal binding scheme was 7.9 μM. Cell exposure to serotonin (10 μM) alone failed to alter IK(DR), while fluoxetine (10 μM), a compound structurally similar to VOR, mildly suppressed current amplitude. In continued presence of VOR, neither further addition of propranolol nor risperidone reversed VOR-mediated inhibition of IK(DR). Increasing VOR concentration not only depressed IK(DR) conductance but also shifted toward the hyperpolarized potential. As the VOR concentration was raised, the recovery of IK(DR) block became slowed. The IK(DR) activated by a downsloping ramp was suppressed by its presence. The inhibition of IK(DR) by a train pulse was enhanced during exposure to VOR. In Neuro-2a cells, this drug decreased IK(DR). Overall, inhibitory effects of VOR on ionic currents might constitute another underlying mechanism of its actions.

Carfilzomib (CFZ, Kyprolis®) is widely recognized as an irreversible inhibitor of proteasome activity; however, its actions on ion currents in electrically excitable cells are largely unresolved. The possible actions of CFZ on ionic currents and membrane potential in pituitary GH3, A7r5 vascular smooth muscle, and heart-derived H9c2 cells were extensively investigated in this study. The presence of CFZ suppressed the amplitude of delayed-rectifier K+ current ( I K(DR)) in a time-, state-, and concentration-dependent manner in pituitary GH3 cells. Based on minimal reaction scheme, the value of dissociation constant for CFZ-induced open-channel block of I K(DR) in these cells was 0.33 µM, which is similar to the IC50 value (0.32 µM) used for its efficacy on inhibition of I K(DR) amplitude. Recovery from I K(DR) block by CFZ (0.3 µM and 1 µM) could be well fitted by single exponential with 447 and 645 ms, respectively. The M-type K+ current, another type of K+ current elicited by low-threshold potential, was slightly suppressed by CFZ (1 µM). Under current-clamp condition, addition of CFZ depolarized GH3 cells, broadened the duration of action potentials as well as raised the firing frequency. In A7r5 vascular smooth muscle cells or H9c2 cardiac cells, the CFZ-induced inhibition of I K(DR) remained efficacious. Therefore, our study led us to reflect that CFZ or other structurally similar compounds should somehow act on the activity of membrane KV channels through which they influence the functional activities in different types of electrically excitable cells such as endocrine, neuroendocrine cells, smooth muscle cells, or heart cells, if similar in vivo findings occur.

Phenobarbital (PHB, Luminal Sodium®) is a medication of the barbiturate and has long been recognized to be an anticonvulsant and a hypnotic because it can facilitate synaptic inhibition in the central nervous system through acting on the γ-aminobutyric acid (GABA) type A (GABAA) receptors. However, to what extent PHB could directly perturb the magnitude and gating of different plasmalemmal ionic currents is not thoroughly explored. In neuroblastoma Neuro-2a cells, we found that PHB effectively suppressed the magnitude of voltage-gated Na+ current (INa) in a concentration-dependent fashion, with an effective IC50 value of 83 µM. The cumulative inhibition of INa, evoked by pulse train stimulation, was enhanced by PHB. However, tefluthrin, an activator of INa, could attenuate PHB-induced reduction in the decaying time constant of INa inhibition evoked by pulse train stimuli. In addition, the erg (ether-à-go-go-related gene)-mediated K+ current (IK(erg)) was also blocked by PHB. The PHB-mediated inhibition on IK(erg) could not be overcome by flumazenil (GABA antagonist) or chlorotoxin (chloride channel blocker). The PHB reduced the recovery of IK(erg) by a two-step voltage protocol with a geometrics-based progression, but it increased the decaying rate of IK(erg), evoked by the envelope-of-tail method. About the M-type K+ currents (IK(M)), PHB caused a reduction of its amplitude, which could not be counteracted by flumazenil or chlorotoxin, and PHB could enhance its cumulative inhibition during pulse train stimulation. Moreover, the magnitude of delayed-rectifier K+ current (IK(DR)) was inhibited by PHB, while the cumulative inhibition of IK(DR) during 10 s of repetitive stimulation was enhanced. Multiple ionic currents during pulse train stimulation were subject to PHB, and neither GABA antagonist nor chloride channel blocker could counteract these PHB-induced reductions. It suggests that these actions might conceivably participate in different functional activities of excitable cells and be independent of GABAA receptors.