Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca²⁺ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca_V1.3L-type Ca²⁺ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca_V1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca_Vα₁ ion-conducting subunit of the Ca_V1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca²⁺ macroscopic currents and impair insulin release stimulated with high K⁺. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca_V1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca_Vα₁ pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca_V1.3 channels and contribute to regulate insulin secretion.     

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens

The dominant role of Ca_V2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of Ca_V2 and Ca_V1 channels, and less so Ca_V3 channels, it is unclear why there have not been major shifts toward dependence on other Ca_V channels for synaptic transmission. Here, we provide a structural and functional profile of the Ca_V2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for Ca_V1–Ca_V3 channels. Remarkably, the highly divergent channel possesses similar features as human Ca_V2.1 and other Ca_V2 channels, including high voltage–activated currents that are larger in external Ba²⁺ than in Ca²⁺; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax Ca_V2 suggests that the core features of presynaptic Ca_V2 channels were established early during animal evolution, after Ca_V1 and Ca_V2 channels emerged via proposed gene duplication from an ancestral Ca_V1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian Ca_V2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd²⁺ and Ni²⁺. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human Ca_V2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) remain unknown. We analyzed mechanisms regulating resting [Ca²⁺]_i in dissociated rat melanotrophs by Ca²⁺-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca²⁺ channels (VGCCs) as well as removal of extracellular Ca²⁺ resulted in a rapid and reversible decrease in [Ca²⁺]_i, indicating constitutive Ca²⁺ influx through L-type VGCCs. Reduction of extracellular Na⁺ concentration (replacement with NMDG⁺) similarly decreased resting [Ca²⁺]_i. When cells were champed at –80 mV, decrease in the extracellular Na⁺ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na⁺ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K⁺ concentration was increased from 5 mM to 50 mM in the presence of K⁺ channel blockers, Ba²⁺ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca²⁺]_i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca²⁺]_i in rat melanotrophs.     

Effects of K+ Channel Blockers and Nitric Oxide on the Electrical and Contractile Activities of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a sucrose-bridge technique, we studied electrical and mechanical responses of smooth muscle ring strips of the rabbit main pulmonary artery to applications of blockers of voltage-operated (including Ca²⁺-dependent) K⁺ channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), as well to application of nitric oxide (NO); nitroglycerin (NG) was used as a donor of the latter. All experiments were carried out under conditions of blockade of the adreno- and cholinoreceptors in the preparation. Both TEA and 4-AP evoked dose-dependent effects: depolarization of smooth muscle cells (SMC) and their contraction. Simultaneous addition of TEA and 4-AP to the normal superfusate (Krebs solution) resulted in intensification of depolarization and initiated generation of action potentials (AP); contractions became rather intensive and possessed a tetanic pattern. Addition of NG to TEA- and 4-AP-containing Krebs solution effectively suppressed AP generation and contractions, whereas the depolarization level underwent only mild modifications. These findings show that Ca²⁺-dependent high-conductance K⁺ channels (K_Ca channels) and 4-AP-sensitive voltage-operated K⁺ channels (K_V channels) are involved in the formation of the resting membrane potential (RMP) in SMC of the rabbit main pulmonary artery. The impact of the K_Ca channels is greater than that of the K_V channels. We suppose that the effects of NO on SMC are related to inhibition of the activity of high-threshold voltage-operated L-type Ca²⁺ channels and, probably, to lowering of the sensitivity of the contractile SMC apparatus to Ca²⁺.     

Distribution of ion channels on taste cells and its relationship to chemosensory transduction

Summary The presence and regional localization of voltagegated ion channels on taste cells inNecturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na⁺, K⁺, or Ca²⁺ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K⁺ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na⁺ and Ca²⁺ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K⁺ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K⁺ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K⁺ conductance may underlie receptor potentials ellicted by rapid stimuli.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Electrical coupling between the human serotonin transporter and voltage-gated Ca channels

Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca²⁺ mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca²⁺ permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca²⁺ channels (Ca_V). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in Ca_V1.1-null muscle cells, thus implicating Ca²⁺ channels. Ca_V1.3 and Ca_V2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type Ca_V1.3 channel show Ca²⁺ transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and Ca_V1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca²⁺ channels such as Ca_V1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca²⁺-driven signals in excitable cells.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration

Epithelial cell migration plays an important role in gastrointestinal mucosal repair. We previously reported that multiple functional ion channels, including a Ba²⁺-sensitive K⁺ inward rectifier K_ir1.2, 4-aminopyridine (4-AP)-sensitive voltage-gated K⁺ channels K_v1.1, K_v1.6 and K_v2.1, and a nifedipine-sensitive, tetrodotoxin (TTX)-insensitive voltage-gated Na⁺ channel Na_v1.5 were expressed in a non-transformed rat gastric epithelial cell line (RGM-1). In the present study, we further investigated whether these ion channels are involved in the modulation of gastric epithelial cell migration. Cell migration was determined by monolayer wound healing assay. Results showed that blockade of K_v with 4-AP or Na_v1.5 with nifedipine inhibited RGM-1 cell migration in the absence or presence of epidermal growth factor (EGF), which effectively stimulated RGM-1 cell migration. Moreover, high concentration of TTX mimicked the action of nifedipine, suggesting that the action of nifedipine was mediated through specific blockade of Na_v1.5. In contrast, inhibition of K_ir1.2 with Ba²⁺, either in basal or EGF-stimulated condition, had no effect on RGM-1 cell migration. In conclusion, the present study demonstrates for the first time that voltage-gated K⁺ and Na⁺ channels are involved in the modulation of gastric epithelial cell migration.     

Exploring the dominant role of Cav1 channels in signalling to the nucleus

Calcium is important in controlling nuclear gene expression through the activation of multiple signal-transduction pathways in neurons. Compared with other voltage-gated calcium channels, Ca_V1 channels demonstrate a considerable advantage in signalling to the nucleus. In this review, we summarize the recent progress in elucidating the mechanisms involved. Ca_V1 channels, already advantaged in their responsiveness to depolarization, trigger communication with the nucleus by attracting colocalized clusters of activated CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Ca_V2 channels lack this ability, but must work at a distance of >1 μm from the Ca_V1-CaMKII co-clusters, which hampers their relative efficiency for a given rise in bulk [Ca²⁺]_i (intracellular [Ca²⁺]). Moreover, Ca²⁺ influx from Ca_V2 channels is preferentially buffered by the ER (endoplasmic reticulum) and mitochondria, further attenuating their effectiveness in signalling to the nucleus.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Mechanisms involved in testosterone-induced relaxation to the pig urinary bladder neck

ObjectivesTestosterone replacement therapy improves bladder capacity in urinary tract dysfunction. There is no information, however, about the role of this steroid hormone on the muscle tension of the bladder outflow region. The current study investigated the mechanisms underlying the testosterone-induced action in the pig bladder neck.MethodsUrothelium-denuded bladder neck strips were mounted in myographs for isometric force recordings and for simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension. The relaxations to testosterone, the non-aromatizable metabolite 4,5α-dihydrotestosterone (DHT) and electrical field stimulation (EFS) were carried out on phenylephrine (PhE)-precontracted strips.ResultsTestosterone and DHT evoked similar concentration-dependent relaxations only at very high pharmacological concentrations. The presence of the urothelium and the inhibition of intracellular androgenic receptor (AR), aromatase, 5α-reductase, nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX), large-, intermediate- and small-Ca²⁺-activated K⁺ channels or ATP-dependent K⁺ channels failed to modify the testosterone relaxations. Neuronal voltage-gated Ca²⁺ (VOC) channels and voltage-gated K⁺ (K_V) channel blockers potentiated these responses. EFS evoked frequency-dependent relaxations, which were not changed by threshold concentrations of testosterone. In Ca²⁺-free potassium rich physiological saline solution, testosterone inhibited the contractions induced by CaCl₂ and the L-type VOC channel activator (±)-BAY K 8644. Relaxations elicited by testosterone were accompanied by simultaneous decreases in smooth muscle [Ca²⁺]_i.ConclusionsTestosterone produces relaxation of the pig urinary bladder neck through mechanisms independent of urothelium, AR, aromatase, 5α-reductase, NO synthase, guanylyl cyclase, COX and K⁺ channels. Testosterone-induced relaxation is produced via the inhibition of the extracellular Ca²⁺ entry through L-type VOC channels.