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BKCa-channel activity often affects the firing properties of neurons, the shapes of neuronal action potentials (APs), and in some cases the extent of neurotransmitter release. It has become clear that BKCa channels often form complexes with voltage-gated Ca2+ channels (CaV channels) such that when a CaV channel is activated, the ensuing influx of Ca2+ activates its closely associated BKCa channel. Thus, in modeling the electrical properties of neurons, it would be useful to have quantitative models of CaV/BKCa complexes. Furthermore, in a population of CaV/BKCa complexes, all BKCa channels are not exposed to the same Ca2+ concentration at the same time. Thus, stochastic rather than deterministic models are required. To date, however, no such models have been described. Here, however, I present a stochastic model of a CaV2.1/BKCa(α-only) complex, as might be found in a central nerve terminal. The CaV2.1/BKCa model is based on kinetic modeling of its two component channels at physiological temperature. Surprisingly, The CaV2.1/BKCa model predicts that although the CaV channel will open nearly every time during a typical cortical AP, its associated BKCa channel is expected to open in only 30% of trials, and this percentage is very sensitive to the duration of the AP, the distance between the two channels in the complex, and the presence of fast internal Ca2+ buffers. Also, the model predicts that the kinetics of the BKCa currents of a population of CaV2.1/BKCa complexes will not be limited by the kinetics of the CaV2.1 channel, and during a train of APs, the current response of the complex is expected to faithfully follow even very rapid trains. Aside from providing insight into how these complexes are likely to behave in vivo, the models presented here could also be of use more generally as components of higher-level models of neural function.

Voltage-gated Ca2+ (Cav) channels play an essential role in the regulation of renal blood flow and glomerular filtration rate (GFR). Because T-type Cav channels are differentially expressed in pre- and postglomerular vessels, it was hypothesized that they impact renal blood flow and GFR differentially. The question was addressed with the use of two T-type Cav knockout (Cav3.1−/− and Cav3.2−/−) mouse strains. Continuous recordings of blood pressure and heart rate, para-aminohippurate clearance (renal plasma flow), and inulin clearance (GFR) were performed in conscious, chronically catheterized, wild-type (WT) and Cav3.1−/− and Cav3.2−/− mice. The contractility of afferent and efferent arterioles was determined in isolated perfused blood vessels. Efferent arterioles from Cav3.2−/− mice constricted significantly more in response to a depolarization compared with WT mice. GFR was increased in Cav3.2−/− mice with no significant changes in renal plasma flow, heart rate, and blood pressure. Cav3.1−/− mice had a higher renal plasma flow compared with WT mice, whereas GFR was indistinguishable from WT mice. No difference in the concentration response to K+ was observed in isolated afferent and efferent arterioles from Cav3.1−/− mice compared with WT mice. Heart rate was significantly lower in Cav3.1−/− mice compared with WT mice with no difference in blood pressure. T-type antagonists significantly inhibited the constriction of human intrarenal arteries in response to a small depolarization. In conclusion, Cav3.2 channels support dilatation of efferent arterioles and affect GFR, whereas Cav3.1 channels in vivo contribute to renal vascular resistance. It is suggested that endothelial and nerve localization of Cav3.2 and Cav3.1, respectively, may account for the observed effects.

Sevoflurane is one of inhalation anesthetics and has been commonly used in obstetric and pediatric anesthesia. The widespread use of sevoflurane in newborns and infants has made its safety a health issue of concern. Voltage-gated Ca(2+) channels (VGCCs) play an important role in neuronal excitability and are essential for normal brain development. However, the role of sevoflurane on regulating Ca(2+) channels during the period of rapid brain development is still not well understood. The aim of this study is to explore the effects of sevoflurane on voltage-gated Ca(2+) channels for hippocampal CA1 pyramidal neurons during the period of rapid brain development. 1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6h). Whole-cell patch clamp technique was used. I-V curve, steady-state activation and inactivation curves of Ca(2+) channels were studied in rats of the both 3 treated groups at 5 different ages (1 week, 2 weeks, 3 weeks, 4 and 5 weeks old). After anesthesia with sevoflurane at 1-week-old rats, Ca(2+) channels current density was significantly decreased at week 1 and week 2 (p

Aim The present study was designed to investigate the possible role of T-type Ca2+ channels and HCN channels in the development of stress adaptation in cold-water immersion stress-subjected mice. Material and methods The mice were subjected to cold-water immersion stress by placing them individually in a water tank (depth = 15.5 cm; temperature = 15 ± 2 °C) for 5 min. The mice were subjected to single episode of cold-water immersion stress for inducing acute stress; while for inducing stress adaptation, mice were subjected to repeated episodes of homotypic stressor (5 min) for 5 consecutive days. Animals were administered with ethosuximide (100 and 200 mg/kg, i.p.) and ivabradine (5 and 10 mg/kg, i.p.) before subjecting them to stress for five days. The stress-related behavioral alterations were assessed using the actophotometer, the hole board, the open field and the social interaction tests. The plasma corticosterone levels were quantified as a biochemical parameter of hypothalamic-pituitary-adrenal (HPA) axis activation. Results Acute stress altered the behavioral and biochemical parameters of the animals. However, repeated stress significantly restored the behavioral and biochemical alterations signifying the development of adaptation. Administration of ethosuximide and ivabradine abolished the restoration of behavioral and biochemical changes in the animals subjected to repeated stress. Conclusion The ethosuximide and ivabradine mediated attenuation of stress adaptation demonstrates that the opening of T-type Ca2+ channels and activation of HCN channels are involved in inducing stress adaptation in repeated stress-subjected animals.

Summary Coupling distances between Ca2+ channels and release sensors regulate vesicular release probability (pv). Tight coupling is thought to provide a framework for high pv and loose coupling for high plasticity at low pv. At synapses investigated during development, coupling distances decrease, thereby increasing pv and transmission fidelity. We find that neocortical high-fidelity synapses deviate from these rules. Paired recordings from pyramidal neurons with “slow” and “fast” Ca2+ chelators combined with experimentally constrained simulations suggest that coupling tightens significantly during development. However, fluctuation analysis revealed that neither pv (∼0.63) nor the number of release sites (∼8) changes concomitantly. Moreover, the amplitude and time course of presynaptic Ca2+ transients are not different between age groups. These results are explained by high-pv release sites with Ca2+ microdomains in young synapses and nanodomains in mature synapses. Thus, at neocortical synapses, a developmental reorganization of the active zone leaves pv unaffected, emphasizing developmental and functional synaptic diversity.

The article was originally published with one author missing. The name of the co-author Roman Moravcik was inadvertently omitted. His name and affiliation have now been added to the author list. The original article has been corrected.

Voltage-gated calcium (CaV) channel, CaV β subunit, phosphatidylinositol 4,5-bisphosphate (PIP2), molecular gating mechanism

We demonstrated the inhibitory effect of NNC 55-0396, a T-type Ca2+ channel inhibitor, on voltage-dependent K+ (KV) channels in freshly isolated rabbit coronary arterial smooth muscle cells. NNC 55-0396 decreased the amplitude of KV currents in a concentration-dependent manner, with an IC50 of 0.080 μM and a Hill coefficient of 0.76. NNC 55-0396 did not affect steady-state activation and inactivation curves, indicating that the compound does not affect the voltage sensitivity of KV channel gating. Both the KV currents and the inhibitory effect of NNC 55-0396 on KV channels were not altered by depletion of extracellular Ca2+ or intracellular ATP, suggesting that the inhibitory effect of NNC 55-0396 is independent of Ca2+-channel activity and phosphorylation-dependent signaling cascades. From these results, we concluded that NNC 55-0396 dose-dependently inhibits KV currents, independently of Ca2+-channel activity and intracellular signaling cascades. Keywords:: NNC 55-0396, voltage-dependent K+ channel, coronary artery

Local Ca(2+) signals through voltage-gated Ca(2+) channels (CaVs) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single CaV channel and the Ca(2+) signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca(2+) measurements. Here, we exploited Ca(2+)-dependent inactivation of CaV channels as a nanometer-range Ca(2+) indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca(2+) amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of CaV nanodomains, as diffusion-restricted environments that amplify small Ca(2+) fluxes into enormous local Ca(2+) concentrations. This Ca(2+) tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca(2+) load.

The effect of efonidipine, a commercially available antihypertensive drug and Ca2 + channel inhibitor, on voltage-dependent K+ (Kv) channels was studied in freshly isolated rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. The amplitude of Kv current was decreased by application of efonidipine in a dose-dependent manner, with IC50 of 0.26 μM and a Hill coefficient of 0.91, which suggests 1:1 binding stoichiometry. Efonidipine did not affect voltage-dependent activation of the Kv channel, but shifted the inactivation curve by − 8.87 mV. The inhibitory effect of efonidipine was not significantly changed by depletion of extracellular Ca2 + or intracellular ATP, which indicated no involvement of the Ca2 + channel or intracellular protein kinase-dependent cascades. We conclude that efonidipine dose-dependently inhibits Kv current in a phosphorylation- and Ca2 + channel-independent manner.

Stochastic modelling of coupled Ca(2+) channels is a challenge, especially when the coupling of the channels, as determined by their spatial arrangement relative to each other, has to be considered at multiple spatial scales. In this study, the authors address this problem using coloured stochastic Petri nets (𝒮𝒫𝒩(𝒸)) as high‐level description to generate continuous‐time Markov chains. The authors develop several models with increasing complexity. They first apply 𝒮𝒫𝒩(𝒸) to model single clusters of coupled Ca(2+) channels arranged in a regular or irregular lattice, where they describe how to represent the geometrical arrangement of Ca(2+) channels relative to each other using colours. They then apply this modelling idea to construct more complex models by modelling spatially arranged clusters of channels. The authors’ models can be easily reproduced and adapted to different scenarios.

Low-voltage-activated T-type Ca2+ channels are highly expressed in the thalamocortical circuit, suggesting that they play a role in this brain circuit. Indeed, low-threshold burst firing mediated by T-type Ca2+ channels has long been implicated in the synchronization of the thalamocortical circuit. Over the past few decades, the conventional view has been that rhythmic burst firing mediated by T-type channels in both thalamic reticular nuclie (TRN) and thalamocortical (TC) neurons are equally critical in the generation of thalamocortical oscillations during sleep rhythms and spike-wave-discharges (SWDs).This review broadly investigates recent studies indicating that even though both TRN and TC nuclei are required for thalamocortical oscillations, the contributions of T-type channels to TRN and TC neurons are not equal in the genesis of sleep spindles and SWDs. T-type channels in TC neurons are an essential component of SWD generation, whereas the requirement for TRN T-type channels in SWD generation remains controversial at least in the GBL model of absence seizures. Therefore, a deeper understanding of the functional consequences of modulating each T-type channel subtype could guide the development of therapeutic tools for absence seizures while minimizing side effects on physiological thalamocortical oscillations. This article is part of a Special Issue entitled: Calcium channels.

Low-voltage-activated T-type Ca2+ channels are widely expressed in various types of neurons. Once deinactivated by hyperpolarization, T-type channels are ready to be activated by a small depolarization near the resting membrane potential and, therefore, are optimal for regulating the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Ca2+ influx through T-type channels engenders low-threshold Ca2+ spikes, which in turn trigger a burst of action potentials. Low-threshold burst firing has been implicated in the synchronization of the thalamocortical circuit during sleep and in absence seizures. It also has been suggested that T-type channels play an important role in pain signal transmission, based on their abundant expression in pain-processing pathways in peripheral and central neurons. In this review, we will describe studies on the role of T-type Ca2+ channels in the physiological as well as pathological generation of brain rhythms in sleep, absence epilepsy, and pain signal transmission. Recent advances in studies of T-type channels in the control of cognition will also be briefly discussed.

Ca(V)1.1 is the prototype for the other nine known Ca(V) channel isoforms, yet it has functional properties that make it truly atypical of this group. Specifically, Ca(V)1.1 is expressed solely in skeletal muscle where it serves multiple purposes; it is the voltage sensor for excitation-contraction coupling and it is an L-type Ca²⁺ channel which contributes to a form of activity-dependent Ca²⁺ entry that has been termed Excitation-coupled Ca²⁺ entry. The ability of Ca(V)1.1 to serve as voltage-sensor for excitation-contraction coupling appears to be unique among Ca(V) channels, whereas the physiological role of its more conventional function as a Ca²⁺ channel has been a matter of uncertainty for nearly 50 years. In this chapter, we discuss how Ca(V)1.1 supports excitation-contraction coupling, the possible relevance of Ca²⁺ entry through Ca(V)1.1 and how alterations of Ca(V)1.1 function can have pathophysiological consequences. This article is part of a Special Issue entitled: Calcium channels.

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca2+ channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca2+ or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K+-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca2+ currents were −2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of −70 mV). Window currents were between −40 and +10 mV with a peak at −15.4 mV. Nifedipine inhibited currents with an IC50 of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K+-mediated increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) due to Ca2+ influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K+ but not in aorta and MA. In contrast, H2O2 inhibited high K+-mediated increase in [Ca2+]cyt and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of CaV1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.

Cerebral blood flow is controlled by a network of resistance arteries that dilate and constrict to mechanical and chemical stimuli. Vasoactive stimuli influence arterial diameter through alterations in resting membrane potential and the influx of Ca2+ through voltage-gated Ca2+ channels. Historically, L-type Ca2+ channels were thought to be solely expressed in cerebral arterial smooth muscle. Recent studies have, however, challenged this perspective, by providing evidence of T-type Ca2+ channels in vascular tissues. This perspective piece will introduce T-type Ca2+ channels, their electrophysiological properties, and potential roles in arterial tone development. We begin with a brief overview of Ca2+ channels and a discussion of the approaches used to isolate this elusive conductance. We will then speculate on how the two T-type Ca2+ channels expressed in cerebral arterial smooth muscle might differentially influence arterial tone. This discovery of T-type Ca2+ channels alters our traditional understanding of Ca2+ dynamics in vascular tissue and fosters new avenues of research and insight into the basis of arterial tone development.

N-type Ca2+channels are densely distributed in sympathetic nerves that innervate renal tubules. However, the role of N-type Ca2+channels in renal fibrosis remains unknown. To address this issue, we examined the difference between the effects of amlodipine (an L-type Ca2+channel blocker) and cilnidipine (a dual L/N-type Ca2+channel blocker) on fibrotic changes using a rat unilateral ureteral obstruction (UUO) model. The expression of both L-type and N-type Ca2+channels was significantly upregulated in UUO kidneys compared with that in contralateral kidneys. There were no significant differences in mean blood pressure among the rats tested. Both amlodipine and cilnidipine significantly attenuated fibrotic changes in UUO kidneys. The antifibrotic effect of cilnidipine was more potent than that of amlodipine. Amlodipine as well as cilnidipine reduced type III collagen deposition, α-smooth muscle actin (α-SMA) expression, and interstitial cell proliferation. In addition, cilnidipine significantly reduced deposition of type I collagen and macrophage infiltration in UUO kidneys. With the use of in vivo bromodeoxyuridine labeling, label-retaining cells (LRCs) were identified as a population of tubular cells that participate in epithelial-mesenchymal transition after UUO. Some LRCs migrated into the interstitium, expressed α-SMA and vimentin, and produced several extracellular matrixes in UUO kidneys. The number of interstitial LRCs was significantly decreased by cilnidipine but not amlodipine. These data suggest that N-type Ca2+channels contribute to multiple steps of renal fibrosis, and its blockade may thus be a useful therapeutic approach for prevention of renal fibrosis.

Symposium on Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest Control / 252nd ACS National Meeting and Exposition, 21-25 aôut 2016 Philadelphia, PA – États-Unis.; International audience; Voltage-gated Ca2+ (CaV) channels allow Ca2+ to enter the cell in response to membrane depolarization. This Ca2+ influx is not only necessary for cell excitability, but also triggers, via Ca2+-binding proteins, important biological functions such as contraction, synaptic transmission, or gene expression. Insect CaV channels are encoded by only three genes (against 10 in mammals), and their invalidation or pharmacological blockade is expected to have deleterious effects. They may thus constitute interesting targets for specific insecticides. However, the precise identification of the genes underlying the different Ca2+ currents recorded in different tissues, as well as the heterologous expression of these genes to screen selective molecules, have been proven to be difficult. This chapter reports on the recent successful expression of honeybee Ca2+ channels genes in Xenopus oocytes and reviews pharmacological properties of Ca2+ currents recorded in isolated honeybee neurons and muscle cells.

Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca2+- and/or stretch-dependent flow-induced K+secretion (FIKS) and contributes to K+adaptation in response to dietary K+loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K+secretory processes. In support of a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore-forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high-K+diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca2+concentration, albeit of nonciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K+-fed rabbits with channel subunit-specific antibodies revealed colocalization of immunodetectable BKα- and β1-subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low-K+vs. high-K+diet. Single-channel recordings of cilia revealed K+channels with conductance and kinetics typical of the BK channel. The observations that 1) FIKS was preserved but 2) the high-amplitude Ca2+peak elicited by flow was reduced in microperfused CCDs subject to pharmacological deciliation suggest that cilia BK channels do not contribute to K+secretion in this segment, but that cilia serve as modulators of cell signaling.

Remote-controlled Ca(2+) influx, elicited by electropotential waves, triggers local signaling cascades in sieve elements and companion cells along the phloem of Vicia faba plants. The stimulus strength seems to be communicated by the rate and duration of Ca(2+) influx into sieve elements (SEs). The cooperative recruitment of Ca(2+) channels results in a graded response of forisome culminating in full sieve-tube occlusion. Several lines of evidence are integrated into a model that links the mode and strength of the electropotential waves (EPWs) with forisome dispersion, mediated by transiently enhanced levels of local Ca(2+) release dependent on both plasma membrane and ER Ca(2+) channels.

Rationale: Cav3.1 T-type calcium channels are expressed during development and then re-expressed in ventricular myocytes after pathological stress such as myocardial infarction (MI). Our lab has previously shown that mice with cardiac specific overexpression of Cav3.1 have improved contractility, survival, and preservation of the myocardial mass post-MI. Hypothesis: Loss of the Cav3.1 subunit of the T-type calcium channel reduces the ability of the heart to adapt to MI due to alterations in the wound healing response. Methods and Results: Ischemia reperfusion (IR) injury was induced in 12-16 week old Cav3.1 -/- and wild type (WT) mice. Previously collected data from our groups showed that Cav3.1 -/- mice have decreased survival after permanent occlusion MI. In the present study there was no significant difference in the one week survival of these mice after IR (100% vs 90% for WT vs Cav3.1 -/- , respectively). Cav3.1 -/- mice had significantly increased heart weight to body weight (7.066 vs 5.452) and heart weight to tibia length ratios (9.284 vs 7.594) compared to WT mice at 7 days post IR. Echocardiography performed at baseline showed no significant differences between the two groups. At 1 week post-IR, Cav3.1 -/- mice had significantly lower ejection fraction (11.58% vs 21.77%) and fractional shortening (10.02% vs 17.46%) compared to WT mice. This decrease in function was accompanied by a significantly increased percentage of fibrosis in the Cav3.1 -/- mice (26.42% vs 11.17%) and this was associated with alterations in the infiltration of proliferative inflammatory cells. The left ventricular internal diameter was also significantly increased in Cav3.1 -/- mice compared to WT (4.766 mm vs 4.151 mm). Summary and Conclusions: Cav3.1 -/- mice have decreased cardiac function and increased pathological remodeling at 1-week post-IR. The mechanisms resulting in these pathological changes appear to involve alterations in post MI inflammatory cell infiltration and myocyte survival. These results suggest that loss of Cav3.1 T-type calcium channels alters the behavior of inflammatory responses and myocyte survival after MI.

Recent findings have revealed a fundamental role of the ubiquitin-proteasome system (UPS) in the regulation of voltage-gated Ca2+ channels (VGCCs). It has been proposed that the ubiquitination-deubiquitination balance dictates the number of channels expressed at the plasma membrane, which in turn influences a number of physiological and pathophysiological processes. This minireview surveys recent studies showing that VGCCs may be ubiquitinated in an unexpectedly complex manner, and highlights the role of the UPS in the regulation of the channels, focusing on the mechanisms that control their cell surface expression. The exciting new findings in this emerging field suggest that the turnover of VGCCs may be determined to a large degree by the activity of the UPS, and that alteration of the UPS molecular machinery may be one of the underlying mechanisms occurring in a number of channelopathies.

Voltage-gated Ca2+ (Cav)1.3 α-subunits of high voltage-activated Ca2+ channels (HVACCs) are essential for Ca2+ influx and transmitter release in cochlear inner hair cells and therefore for signal transmission into the central auditory pathway. Their absence leads to deafness and to striking structural changes in the auditory brain stem, particularly in the lateral superior olive (LSO). Here, we analyzed the contribution of various types of HVACCs to the total Ca2+ current ( ICa) in developing mouse LSO neurons to address several questions: do LSO neurons express functional Cav1.3 channels? What other types of HVACCs are expressed? Are there developmental changes? Do LSO neurons of Cav1.3−/− mice show any compensatory responses, namely, upregulation of other HVACCs? Our electrophysiological and pharmacological results showed the presence of functional Cav1.3 and Cav1.2 channels at both postnatal days 4 and 12. Aside from these L-type channels, LSO neurons also expressed functional P/Q-type, N-type, and, most likely, R-type channels. The relative contribution of the four different subtypes to ICa appeared to be 45%, 29%, 22%, and 4% at postnatal day 12, respectively. The physiological results were flanked and extended by quantitative RT-PCR data. Altogether, LSO neurons displayed a broad repertoire of HVACC subtypes. Genetic ablation of Cav1.3 resulted in functional reorganization of some other HVACCs but did not restore normal ICa properties. Together, our results suggest that several types of HVACCs are of functional relevance for the developing LSO. Whether on-site loss of Cav1.3, i.e., in LSO neurons, contributes to the recently described malformation of the LSO needs to be determined by using tissue-specific Cav1.3−/− animals.

To observe the relaxant effect of Aike Mixture (AKM) on isolated bladder and prostatic urethral smooth muscle of rabbits.The isolated bladder and prostatic urethral smooth muscle from male rabbits were placed in a Magnus bath and smooth muscle contraction was measured using a biological signal acquisition and analysis system. The effects of AKM in combination with methoxyamine, carbachol and CaCl2 on the contractile tension of muscle strips were determined by cumulative dosing.AKM dose-dependently reduced contractile tension of bladder trigone smooth muscle (r=0.831, P0.05), reduced contractile wave amplitude (r=0.837, P0.05) and decreased contractile frequency (r=-0.917, P0.01). AKM significantly inhibited the increases in smooth muscle contraction induced by methoxyamine, carbachol and CaCl2.AKM dose-dependently inhibited the contraction of rabbit isolated bladder and prostatic urethral smooth muscle by antagonizing α1-adrenergic receptors and M-cholinergic receptors.

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC β(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.

The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nucleated erythrocytes. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nucleocytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are manifested as a decrease of the cell volume. The blocker of Ca2+-channels verapamil produces a transitory increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nucleocytoplasmic transfer.

Ca2+ currents ( ICa) recorded from adrenal chromaffin cells (CCs) of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats are similar to one another, but different from those recorded in other rodent species. ICa in WKY/SHR CCs comprises an early, transient ( ICae) and a late, sustained component ( ICas). In Wistar CCs, ICae is absent, and ICas is of greater amplitude. Activation and steady-state inactivation of ICae and ICas in WKY/SHR CCs suggest the recruitment of at least two populations of Ca2+ channels with different voltage dependence and kinetics. In WKY/SHR CCs, ICae is inhibited by nifedipine, enhanced by BAY K 8644, is not blocked by the mibefradil analog NNC 55–0396, and displays Ca2+-dependent inactivation and fast deactivation kinetics, suggesting that it results from the opening of L-type rather than T-type Ca2+ channels. ICae properties suggest that it originates from the opening of Ca2+ channels formed with the short splice variant (CaV1.342A). RT-PCR showed that expression of CaV1.342A mRNA is similar in both Wistar and WKY/SHR, but that the long variant (CaV1.342) is virtually absent in WKY/SHR. Thus ICae corresponds to the recruitment of CaV1.342A channels, unmasked by the absence of CaV1.342 channels. Studies in WKY CCs do not report major functional alterations, despite the unusual expression pattern of CaV1.3 splice variants. It remains to be established if more subtle functional alterations exist, and if the atypical splicing pattern of CaV1.3 could be related to the functional and behavioral alterations reported in WKY/SHR rats, including their susceptibility to develop hypertension.

Endostatin, a fragment of collagen XVIII, is known as an endogenous angiogenesis inhibitor, and its serum concentration increases in various cardiovascular diseases. T-type Ca2+ channel, low voltage-activated Ca2+ channel, is not expressed in adult ventricular myocytes. Re-expression of T-type Ca2+ channels in cardiac myocytes is thought to be involved in the development of cardiac hypertrophy. We examined the effects of endostatin on T-type Ca2+ channel current by whole-cell patch clamp technique in freshly isolated adult guinea pig ventricular myocytes, which exceptionally express T-type Ca2+ channels. Although endostatin 300 ng/ml had no effect on L-type Ca2+ current, it significantly inhibited T-type Ca2+ current. These data indicate that endostatin can be an endogenous inhibitor of T-type Ca2+ channels in the cardiac myocytes.

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca2+-activated potassium channels (KCa2), specifically isoforms KCa2.2 and 2.3, may control these contractions through negative feedback regulation of Ca2+ entry. We tested whether selective pharmacologic activation of KCa2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both KCa2.2 and KCa2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the KCa2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective KCa2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC50 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca2+ events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F2α did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial KCa2.2/2.3 channels effectively suppresses Ca2+-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.

The modulation of voltage-gated calcium channels by chlorantraniliprole in the central neurones isolated from third-instar larvae of Spodoptera exigua is studied by the whole-cell patch-clamp technique. The current of calcium in the third-instar larvae of S. exigua is identified as a high-voltage activated Ca2+ current. During the 10-min recording, the current–voltage relationship curves of whole-cell calcium channels are shifted in a negative direction by 10 mV compared with the control group. The fact that the gravity rundown of calcium current in the treated group is more apparent than in the control group demonstrates that the open channels are constantly inactivated. In addition, chlorantraniliprole inhibits the recorded calcium currents in a concentration-dependent manner, which is irreversible on washout.

Neurons communicate through Ca2+-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales. SIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.

Synapse formation is tightly associated with neuronal excitability. We found striking synaptic overgrowth caused byDrosophilaK+-channel mutations of theseizureandslowpokegenes, encoding Erg and Ca2+-activated large-conductance (BK) channels, respectively. These mutants display two distinct patterns of “satellite” budding from larval motor terminus synaptic boutons. Double-mutant analysis indicates that BK and Erg K+channels interact with separate sets of synaptic proteins to affect distinct growth steps. Post-synaptic L-type Ca2+channels, Dmca1D, and PSD-95-like scaffold protein, Discs large, are required for satellite budding induced byslowpokeandseizuremutations. Pre-synapticcacophonyCa2+channels and the NCAM-like adhesion molecule, Fasciclin II, take part in a maturation step that is partially arrested byseizuremutations. Importantly,slowpokeandseizuresatellites were both suppressed byrutabagamutations that disrupt Ca2+/CaM-dependent adenylyl cyclase, demonstrating a convergence of K+channels of different functional categories in regulation of excitability-dependent Ca2+influx for triggering cAMP-mediated growth plasticity.

Modifications in Ca(2+) influx may lead to profound changes in the cell activity associated with Ca(2+)-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca(2+) channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca(2+) concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.

Burst firing mediated by a low-threshold spike (LTS) is the hallmark of many thalamic neurons. However, postburst afterhyperpolarizations (AHPs) are relatively uncommon in thalamus. We now report data from patch-clamp recordings in rat brain slice preparations that reveal an LTS-induced slow AHP (sAHP) in thalamic paraventricular (PVT) and other midline neurons, but not in ventrobasal or reticular thalamic neurons. The LTS-induced sAHP lasts 8.9 ± 0.4 s and has a novel pharmacology, with resistance to tetrodotoxin and cadmium and reduction by Ni2+ or nominally zero extracellular calcium concentration, which also attenuate both the LTS and sAHP. The sAHP is inhibited by 10 mM intracellular EGTA or by equimolar replacement of extracellular Ca2+ with Sr2+, consistent with select activation of LVA T-type Ca2+ channels and subsequent Ca2+ influx. In control media, the sAHP reverses near EK+, shifting to −78 mV in 10.1 mM [K+]o and is reduced by Ba2+ or tetraethylammonium. Although these data are consistent with opening of Ca2+-activated K+ channels, this sAHP lacks sensitivity to specific Ca2+-activated K+ channel blockers apamin, iberiotoxin, charybdotoxin, and UCL-2077. The LTS-induced sAHP is suppressed by a β-adrenoceptor agonist isoproterenol, a serotonin 5-HT7 receptor agonist 5-CT, a neuropeptide orexin-A, and by stimulation of the cAMP/protein kinase A pathway with 8-Br-cAMP and forskolin. The data suggest that PVT and certain midline thalamic neurons possess an LTS-induced sAHP that is pharmacologically distinct and may be important for information transfer in thalamic–limbic circuitry during states of attentiveness and motivation.

Low voltage-activated, rapidly inactivating T-type Ca2+ channels are found in a variety of cells, where they regulate electrical activity and Ca2+ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu2+) inhibited T-type Ca2+ current (I T-Ca) with IC50 of 12.06 μM. Inhibition of I T-Ca by Cu2+ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu2+ (10 μM) inhibited I T-Ca by 49.6 ± 4.1%. Inhibition of I T-Ca by Cu2+ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu2+ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca2+ channels, we analyzed the effect of Cu2+ on activation, inactivation, deactivation and reactivation of T-type Ca2+ channels. Since T-type Ca2+ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu2+ can affect male reproductive function through T-type Ca2+ channels as a preconception contraceptive material.

The largest peripheral blood pressure drop occurs in terminal arterioles (2+ channels (VDCCs) are considered the primary pathway for Ca2+ influx during physiologic activation of vascular smooth muscle cells (VSMC). Recent evidence suggests that T-type VDCCs are expressed in renal afferent and efferent arterioles, mesenteric arterioles, and skeletal muscle arterioles. T-type channels are small-conductance, low voltage-activated, fast-inactivating channels. Thus, their role in supplying Ca2+ for contraction of VSMC has been disputed. However, T-type channels display non-inactivating window currents, which may play a role in sustained Ca2+ entry. Here, we review the possible role of T-type channels in vasomotor tone regulation in rat mesenteric terminal arterioles. The CaV3.1 channel was immunolocalized in VSMC, whereas the CaV3.2 channel was predominantly expressed in endothelial cells. Voltage-dependent Ca2+ entry was inhibited by the new specific T-type blockers R(–)-efonidipine and NNC 55-0396. The effect of NNC 55-0396 persisted in depolarized arterioles, suggesting an unusually high activation threshold of mesenteric T-type channels. T-type channels were not necessary for conduction of vasoconstriction, but appear to be important for local electromechanical coupling in VSMC. The first direct demonstration of endothelial T-type channels warrants new investigations of their role in vascular biology.

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine KCa channel blockers is described and their selectivity against KCa channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.

Transient elevations in cytoplasmic Ca(2+) trigger a multitude of Ca(2+)-dependent processes in CNS neurons and many other cell types. The specificity, speed, and reliability of these processes is achieved and ensured by tightly restricting Ca(2+) signals to very local spatiotemporal domains, "Ca(2+) nano- and microdomains," that are centered around Ca(2+)-permeable channels. This arrangement requires that the Ca(2+)-dependent effectors reside within these spatial boundaries where the properties of the Ca(2+) domain and the Ca(2+) sensor of the effector determine the channel-effector activity. We use Ca(2+)-activated K(+) channels (K(Ca)) with either micromolar (BK(Ca) channels) or submicromolar (SK(Ca) channels) affinity for Ca(2+) ions to provide distance constraints for Ca(2+)-effector coupling in local Ca(2+) domains and review their significance for the cell physiology of K(Ca) channels in the CNS. The results may serve as a model for other processes operated by local Ca(2+) domains.

In T-type Ca(2+) channels, macroscopic I(Ba) is usually smaller than I(Ca), but at high Ca(2+) and Ba(2+), single-channel conductance (gamma) is equal. We investigated gamma as a function of divalent concentration and compared it to macroscopic currents using Ca(V)3.1 channels studied under similar experimental conditions (TEA(o) and K(i)). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent gamma was underestimated at depolarized voltages. To estimate divalent gamma, concentration dependence, i(Div), was measured at voltages-50 mV. Data were well described by Langmuir isotherms with gamma(max)(Ca(2+)) of 9.5 +/- 0.4 pS and gamma(max)(Ba(2+)) of 10.3 +/- 0.5 pS. Apparent K(M) was lower for Ca(2+) (2.3 +/- 0.7 mM) than for Ba(2+) (7.9 +/- 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca(2+). As predicted by gamma, macroscopic G(maxCa) was larger than G(maxBa) at 5 mM (G(max)Ca(2+)/Ba:(2+)1.43 +/- 0.14) and similar at 60 mM (G(max)Ca(2+)/Ba:(2+)1.10 +/- 0.02). However, over the range of activation, I(Ca) was larger than I(Ba) under both conditions. This was a consequence of the fact that V(rev) was more negative for I(Ba) than for I(Ca), so that the driving force determining I(Ba) was smaller than that determining I(Ca) over the range of potentials in standard current-voltage relationships.