Voltage-activated Ca2 Research Articles

Distinct roles of Drosophila cacophony and Dmca1D Ca2+ channels in synaptic homeostasis: Genetic interactions with slowpoke Ca2+‐activated BK channels in presynaptic excitability and postsynaptic response

Ca(2+) influx through voltage-activated Ca(2+) channels and its feedback regulation by Ca(2+) -activated K(+) (BK) channels is critical in Ca(2+) -dependent cellular processes, including synaptic transmission, growth and homeostasis. Here we report differential roles of cacophony (CaV 2) and Dmca1D (CaV 1) Ca(2+) channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well-established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K(+) current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. We demonstrate contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre- vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo-triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca(2+) channels in slo-triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo-induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca(2+) channels differentially contribute to functional and structural aspects of slo-induced synaptic modifications.

Phosphoinositide substrates of myotubularin affect voltage-activated Ca2+ release in skeletal muscle

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E–C coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P2 being the most effective, leading to an almost complete disappearance of Ca2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca2+ release are inherently maintained at a low level, inefficient on Ca2+ release in normal conditions.

Association between genetic polymorphisms in Ca(v)2.3 (R-type) Ca2+ channels and fentanyl sensitivity in patients undergoing painful cosmetic surgery.

Individual differences in the sensitivity to fentanyl, a widely used opioid analgesic, lead to different proper doses of fentanyl, which can hamper effective pain treatment. Voltage-activated Ca2+ channels (VACCs) play a crucial role in the nervous system by controlling membrane excitability and calcium signaling. Cav2.3 (R-type) VACCs have been especially thought to play critical roles in pain pathways and the analgesic effects of opioids. However, unknown is whether single-nucleotide polymorphisms (SNPs) of the human CACNA1E (calcium channel, voltage-dependent, R type, alpha 1E subunit) gene that encodes Cav2.3 VACCs influence the analgesic effects of opioids. Thus, the present study examined associations between fentanyl sensitivity and SNPs in the human CACNA1E gene in 355 Japanese patients who underwent painful orofacial cosmetic surgery, including bone dissection. We first conducted linkage disequilibrium (LD) analyses of 223 SNPs in a region that contains the CACNA1E gene using genomic samples from 100 patients, and a total of 13 LD blocks with 42 Tag SNPs were observed within and around the CACNA1E gene region. In the preliminary study using the same 100 genomic samples, only the rs3845446 A/G SNP was significantly associated with perioperative fentanyl use among these 42 Tag SNPs. In a confirmatory study using the other 255 genomic samples, this SNP was also significantly associated with perioperative fentanyl use. Thus, we further analyzed associations between genotypes of this SNP and all of the clinical data using a total of 355 samples. The rs3845446 A/G SNP was associated with intraoperative fentanyl use, 24 h postoperative fentanyl requirements, and perioperative fentanyl use. Subjects who carried the minor G allele required significantly less fentanyl for pain control compared with subjects who did not carry this allele. Although further validation is needed, the present findings show the possibility of the involvement of CACNA1E gene polymorphisms in fentanyl sensitivity.

Nesfatin‐1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²⁺ signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²⁺ in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²⁺ concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²⁺ rise is critically dependent on Ca²⁺ influx via P/Q-type voltage-activated Ca²⁺ channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²⁺ by promoting Ca²⁺ influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone.

Expression of the 2-Subunit Distinguishes Synaptic and Extrasynaptic GABAA Receptors in NG2 Cells of the Hippocampus

NG2 cells are equipped with transmitter receptors and receive direct synaptic input from glutamatergic and GABAergic neurons. The functional impact of these neuron-glia synapses is still unclear. Here, we combined functional and molecular techniques to analyze properties of GABA(A) receptors in NG2 cells of the juvenile mouse hippocampus. GABA activated slowly desensitizing responses in NG2 cells, which were mimicked by muscimol and inhibited by bicuculline. To elucidate the subunit composition of the receptors we tested its pharmacological properties. Coapplication of pentobarbital, benzodiazepines, and zolpidem all significantly increased the GABA-evoked responses. The presence of small tonic currents indicated the presence of extrasynaptic GABA(A) receptors. To further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of NG2 cells. The subunits α1, α2, β3, γ1, and γ2 were most abundantly expressed, matching properties resulting from pharmacological characterization. Importantly, lack of the γ2-subunit conferred a high Zn²⁺ sensitivity to the GABA(A) receptors of NG2 cells. Judging from the zolpidem sensitivity, postsynaptic GABA(A) receptors in NG2 cells contain the γ2-subunit, in contrast to extrasynaptic receptors, which were not modulated by zolpidem. To determine the effect of GABA(A) receptor activation on membrane potential, perforated patch recordings were obtained from NG2 cells. In the current-clamp mode, GABA depolarized the cells to approximately -30 mV, indicating a higher intracellular Cl⁻ concentration (∼50 mM) than previously reported. GABA-induced depolarization in NG2 cells might trigger Ca²⁺ influx through voltage-activated Ca²⁺ channels.

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca2+ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca2+ selective filters of all high-voltage-activated Ca2+ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca2+ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Cav3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Cav3.2, including relative permeability between Ba2+ and Ca2+, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd2+ blocking sensitivity, and pH effects, in distinctive ways.

Neurotrophin receptor p75 mediates the uptake of the amyloid beta (Aβ) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons.

A fascinating yet perhaps overlooked trait of the p75 neurotrophin receptor (p75(NTR)) is its ability to bind ligands with no obvious neurotrophic function. Using cultured basal forebrain (BF) neurons, this study demonstrates selective internalization of amyloid β (Aβ) 1-42 in conjunction with p75(NTR) (labelled with IgG192-Cy3) by cholinergic cells. Active under resting conditions, this process was enhanced by high K(+) stimulation and was insensitive to inhibitors of regulated synaptic activity-tetrodotoxin or botulinum neurotoxins (BoNT type/A and/B). Blockade of sarco-endoplasmic reticulum (SERCA) Ca(2+) ATPase with thapsigargin and CPA or chelation of Ca(2+) with EGTA-AM strongly suppressed the endocytosis of p75(NTR), implicating the role of ER released Ca(2+). The uptake of IgG192-Cy3 was also reduced by T-type Ca(2+) channel blocker mibefradil but not Cd(2+), an indiscriminate blocker of high voltage-activated Ca(2+) currents. A strong co-localization of IgG192-Cy3 with late endosome (Rab7) or lysosome (Lamp1) qualifier proteins suggest these compartments as the primary destination for internalized IgG192 and Aβ. Selective uptake and labeling of BF cholinergic cells with IgG192-Cy3 injected into the prefrontal cortex was verified also in vivo. The significance of these findings in relation to Aβ clearance in the cerebral cortex and pathophysiology of Alzheimer's disease is discussed.

Major contribution of sarcoplasmic reticulum Ca2+ depletion during long-lasting activation of skeletal muscle

Depolarization of skeletal muscle fibers induces sarcoplasmic reticulum (SR) Ca2+ release and contraction that progressively decline while depolarization is maintained. Voltage-dependent inactivation of SR Ca2+ release channels and SR Ca2+ depletion are the two processes proposed to explain the decline of SR Ca2+ release during long-lasting depolarizations. However, the relative contribution of these processes, especially under physiological conditions of activation, is not clearly established. Using Fura-2 and Fluo-5N to monitor cytosolic and SR Ca2+ changes, respectively, in voltage-controlled mouse muscle fibers, we show that 2-min conditioning depolarizations reduce voltage-activated cytosolic Ca2+ signals with a V1/2 of −53 mV but also induce SR Ca2+ depletion that decreased the releasable pool of Ca2+ with the same voltage sensitivity. In contrast, measurement of SR Ca2+ changes indicated that SR Ca2+ release channels were inactivated after SR had been depleted and in response to much higher depolarizations with a V1/2 of −13 mV. In response to trains of action potentials, cytosolic Ca2+ signals decayed with time, whereas SR Ca2+ changes remained stable over 1-min stimulation, demonstrating that SR Ca2+ depletion is exclusively responsible for the decline of SR Ca2+ release under physiological conditions of excitation. These results suggest that previous studies using steady-state inactivation protocols to investigate the voltage dependence of Ca2+ release inactivation in fact probed the voltage dependence of SR Ca2+ depletion, and that SR Ca2+ depletion is the only process that leads to Ca2+ release decline during continuous stimulation of skeletal muscle.

Ligand-Gating by Ca2+Is Rate Limiting for Physiological Operation of BKCaChannels

Large conductance Ca(2+)- and voltage-activated potassium channels (BKCa) shape neuronal excitability and signal transduction. This reflects the integrated influences of transmembrane voltage and intracellular calcium concentration ([Ca(2+)]i) that gate the channels. This dual gating has been mainly studied as voltage-triggered gating modulated by defined steady-state [Ca(2+)]i, a paradigm that does not approximate native conditions. Here we use submillisecond changes of [Ca(2+)]i to investigate the time course of the Ca(2+)-triggered gating of BKCa channels expressed in Chinese hamster ovary cells at distinct membrane potentials in the physiological range. The results show that Ca(2+) can effectively gate BKCa channels and that Ca(2+) gating is largely different from voltage-driven gating. Most prominently, Ca(2+) gating displays a pronounced delay in the millisecond range between Ca(2+) application and channel opening (pre-onset delay) and exhibits slower kinetics across the entire [Ca(2+)]i-voltage plane. Both characteristics are selectively altered by co-assembled BKβ4 or an epilepsy-causing mutation that either slows deactivation or speeds activation and reduces the pre-onset delay, respectively. Similarly, co-assembly of the BKCa channels with voltage-activated Ca(2+) (Cav) channels, mirroring the native configuration, decreased the pre-onset delay to submillisecond values. In BKCa-Cav complexes, the time course of the hyperpolarizing K(+)-current response is dictated by the Ca(2+) gating of the BKCa channels. Consistent with Cav-mediated Ca(2+) influx, gating was fastest at hyperpolarized potentials, but decreased with depolarization of the membrane potential. Our results demonstrate that under experimental paradigms meant to approximate the physiological conditions BKCa channels primarily operate as ligand-activated channels gated by intracellular Ca(2+) and that Ca(2+) gating is tuned for fast responses in neuronal BKCa-Cav complexes.

Calcium responses of circadian pacemaker neurons of the cockroach Rhyparobia maderae to acetylcholine and histamine

The accessory medulla (aMe) is the pacemaker that controls circadian activity rhythms in the cockroach Rhyparobia maderae. Not much is known about the classical neurotransmitters of input pathways to the cockroach circadian system. The circadian pacemaker center receives photic input from the compound eye, via unknown excitatory and GABAergic inhibitory entrainment pathways. In addition, neuropeptidergic inputs couple both pacemaker centers. A histamine-immunoreactive centrifugal neuron connects the ventral aMe with projection areas in the lateral protocerebrum and may provide non-photic inputs. To identify neurotransmitters of input pathways to the circadian clock with Fura-2-dependent Ca(2+) imaging, primary cell cultures of the adult aMe were stimulated with acetylcholine (ACh), as the most prominent excitatory, and histamine, as common inhibitory neurotransmitter. In most of aMe neurons, ACh application caused dose-dependent increases in intracellular Ca(2+) levels via ionotropic nicotinic ACh receptors. These ACh-dependent rises in Ca(2+) were mediated by mibefradil-sensitive voltage-activated Ca(2+) channels. In contrast, histamine application decreased intracellular Ca(2+) levels in only a subpopulation of aMe cells via H2-type histamine receptor chloride channels. Thus, our data suggest that ACh is part of the light entrainment pathway while histamine is involved in a non-photic input pathway to the ventral circadian clock of the Madeira cockroach.

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

Sarcoplasmic Reticulum (SR) Ca2+ Depletion Predominantly Contributes to the Decline of SR Ca2+ Release during Long-Lasting Depolarizations in Skeletal Muscle Fibers

High amplitude depolarizations of skeletal muscle fibers induce massive SR Ca2+ release that progressively declines with time and completely annihilates if depolarization is maintained during several tens of seconds. Several processes may be involved in the decline of SR Ca2+ release: cytosolic Ca2+-dependent closure of SR Ca2+ release channels, voltage-dependent inactivation of SR Ca2+ release channels and SR Ca2+ depletion. This study aimed to determine how these processes are involved during long-lasting depolarizations of isolated mouse muscle fibers under voltage control by measuring cytosolic Ca2+ changes using fura-2 or luminal SR Ca2+ changes using fluo5N in the presence of 50 mM internal EGTA. Decays of cytosolic Ca2+ signals elicited by 50-s duration depolarizations became more marked and faster with depolarization amplitude. Pre-depolarizations of 2-min duration and of increasing amplitude induced a reduction of voltage-activated cytosolic Ca2+ signals with a mean voltage of −50 mV inducing half-maximum reduction. A comparable protocol applied to fibers loaded with fluo5N showed that low voltage depolarizing prepulses induced a marked SR Ca2+ depletion that contributed to reduce a subsequent voltage-activated SR Ca2+ change with a mean voltage of −50 mV inducing half-maximum reduction. Measuring SR Ca2+ changes in response to long-lasting depolarizations indicated that SR Ca2+ release channels inactivated in response to much higher depolarizations with a mean half-maximum inactivation voltage of −20 mV. Finally, trains of action potential of 50 s duration produced cytosolic Ca2+ signals that decayed with time, whereas SR Ca2+ changes did not display any sign of inactivation. These results indicate that the decline in SR Ca2+ release during long-lasting depolarizations mainly results from SR Ca2+ depletion. The work was supported by AFM, CNRS and University Lyon 1.

Voltage-Gated Calcium Channels in Neurons Derived from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Voltage-gated calcium channels couple pre-synaptic action potentials to transmitter release in neurons. In this study, we characterized voltage-activated Ca2+ channels expressed in neurons differentiated from human embryonic stem cell (hESC) or induced pluripotent stem cell (iPSC)-derived long-term self-renewing neuroepithelial-like stem cells (lt-NES) at one to three months of differentiation in vitro. Using barium (Ba2+) as the charge carrier, activation and inactivation of voltage-dependent calcium channels were investigated by the whole-cell configuration of the patch-clamp technique. In lt-NES-derived neurons, we found both non-inactivating high-voltage-activated (HVA) and inactivating low-voltage-activated (LVA) Ca2+ currents. Pharmacological characterization of high-threshold Ca2+ currents was attempted using nifedipine (10 μM ), ω-conotoxin-GVIA (ω-CgTx GVIA, 3μM) and ω-agatoxin-IVA (ω-Aga IVA, 200 nM), which block L, N, and P/Q-type Ca2+ currents, respectively. By analysing the averaged current in response to a square depolarizing voltage step protocol from a holding potential of −110 mV to 0 mV in the presence of these specific blockers, we could show that lt-NES cell-derived neurons express N and P/Q-type high-threshold Ca2+ channels. We also observed Ba2+ currents activated at voltages below −40 mV that were sensitive to NiCl2 (100 μM), indicating that T-type Ca2+ channels also contribute to Ca2+ currents in these neurons. Furthermore, the pharmacology of calcium channels involved in synaptic transmission was analyzed with specific agonists and antagonists of different high-voltage-activated calcium channels. Bath application of ω-CgTx GVIA (3μM) or ω-Aga IVA (200 nM) reduced the frequency of spontaneous postsynaptic currents (sPSCs). Thus, neurons differentiated from lt-NES express both high-threshold and low-threshold voltage-gated Ca2+ channels, and both N and P/Q-type Ca2+ channels mediate synaptic transmission in these neurons. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Voltage-Gated Calcium Channels Exist and Function as Fragment-Channels in Neuronal Plasma Membranes. Ioannis E. Michailidis, Kathryn Abele, Wei K. Zhang, Lawrence Geyman, Bochao Lin, Yong Yu, Eftychios Pnevmatikakis, Jian Yang. Biophys. J. 104(2)s: 462a. Full-Text PDF Open Archive

Calcium Transients in Muscle Fibers Expressing Voltage-Sensitive Phosphoinositide Phosphatases

Phosphoinositides play a role in a variety of cellular signaling processes; PtdIns(4,5)P2 regulates the function of several types of ion channels in the plasma membrane and is the source of important second messengers. We tested the possible role of changes in the PtdIns(4,5)P2 level in E-C coupling and Ca2+ homeostasis in mouse muscle fibers under voltage-clamp conditions. Measurements of indo-1 Ca2+ transients in fibers injected with PtdIns(4,5)P2 revealed no significant changes in the voltage-dependence and maximum value for peak Ca2+ release. We then studied the consequences of the activation of a voltage-sensitive PtdIns-phosphatase (VSP). Expression of either Ci-VSP or Dr-VSP was achieved by in vivo electroporation. Confocal images of N-terminally EGFP-tagged Dr-VSP revealed a double-banded pattern of expression consistent with the triadic region and membrane current measurements from a depolarized holding potential showed the presence of a charge movement component consistent with the voltage-sensitive domain active in the t-tubule membrane. Rhod-2 Ca2+ transients generated by single depolarizing pulses within the voltage-dependent range of activation of Ca2+ release appeared unaffected by the presence of either VSP. In order to strongly activate the VSPs, fibers were depolarized by 10 successive 200 ms-long pulses from −80 mV to +80 mV; Ca2+ transients were compared to the ones elicited by an analogous series of pulses to +10 mV: on average, in control and Ci-VSP-positive fibers, the value for peak Ca2+ transient in response to the 10th pulse to +80 mV was 94 ± 7 % (n=9) and 72 ± 6 % (n=7) the value in response to the corresponding pulse to +10 mV, respectively. The significant depression in Ci-VSP expressing fibers may be indicative that depletion of t-tubule PtdIns(4,5)P2 can affect voltage-activated Ca2+ release. The work was supported by AFM, CNRS and Universite Claude Bernard.

Dopamine Release Suppression Dependent on an Increase of Intracellular Ca<sup>2+</sup> Contributed to Rotenone-induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson’s disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca2+ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca2+ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca2+ through Ca2+ influx by opening of the voltage-gated Ca2+ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca2+ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca2+ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.

Multiple Pathways for High Voltage‐Activated Ca2+ Influx in Anterior Pituitary Lactotrophs and Somatotrophs

The present study demonstrates that a significant proportion of high voltage-activated (HVA) Ca(2+) influx in native rat anterior pituitary cells is carried through non-L-type Ca(2+) channels. Using whole-cell patch-clamp recordings and specific Ca(2+) channel toxin blockers, we show that approximately 35% of the HVA Ca(2+) influx in somatotrophs and lactotrophs is carried through Ca(v) 2.1, Ca(v) 2.2 and Ca(v) 2.3 channels, and that somatotrophs and lactotrophs share similar proportions of these non-L-type Ca(2+) channels. Furthermore, experiments on mixed populations of native anterior pituitary cells revealed that the fraction of HVA Ca(2+) influx carried through these non-L-type Ca(2+) channels might even be higher (approximately 46%), suggesting that non-L-type channels exist in the majority of native anterior pituitary cells. Using western blotting, immunoblots for α(1C) , α(1D) , α(1A) , α(1B) and α(1E) Ca(2+) channel subunits were identified in native rat anterior pituitary cells. Additionally, using reverse transcriptase-polymerase chain reaction, cDNA transcripts for α(1C) , α(1D) , α(1A) and α(1B) Ca(2+) channel subunits were identified. Transcripts for α(1E) were nonspecific and transcripts for α(1S) were not detected at all (control). Taken together, these results clearly demonstrate the existence of multiple HVA Ca(2+) channels in the membrane of rat native anterior pituitary cells. Whether these channels are segregated among different membrane compartments was investigated further in flotation assays, demonstrating that Ca(v) 2.1, Ca(v) 1.2 and caveolin-1 were mostly localised in light fractions of Nycodenz gradients (i.e. in lipid raft domains). Ca(v) 1.3 channels were distributed among both light and heavy fractions of the gradients (i.e. among raft and nonraft domains), whereas Ca(v) 2.2 and Ca(v) 2.3 channels were distributed mostly among nonraft domains. In summary, in the present study, we demonstrate multiple pathways for HVA Ca(2+) influx through L-type and non-L-type Ca(2+) channels in the membrane of native anterior pituitary cells. The compartmentalisation of these channels among raft and nonraft membrane domains might be essential for their proper regulation by separate receptors and signalling pathways.

Harmaline Attenuates Voltage - Sensitive Ca2+ Currents in Neurons of the Inferior Olive

Harmaline is one member of a class of tremorgenic harmala alkaloids that have been implicated in neuroprotective effects and neurodegenerative disorders. It has been reported to interact with several neurotransmitter receptors as well as ion exchangers and voltage-sensitive channels. One site of harmaline action in the brain is the inferior olive (IO). Either local or systemic harmaline injection has been reported to increase spiking rate and coherence in the inferior olive and this activation is thought to produce tremor and ataxia through inferior olivary neuron activation of target neurons in the cerebellum, but the cellular mechanism is not yet known. Here, we have performed whole cell voltage-clamp and current clamp recordings from olivary neurons in brain slices derived from newborn rats. We found that both transient low-voltage activated (LVA) and sustained high voltage-activated (HVA) Ca(2+) currents are significantly attenuated by 0.125 - 0.25 mM harmaline applied to the bath and that this attenuation is partially reversible. In current clamp recordings, spike-afterhyperpolarization complexes were evoked by brief positive current injections. Harmaline produced a small attenuation of spike amplitude, but large spike broadening associated with attenuation of the fast and medium afterhyperpolarization. Our data suggest that one mode of olivary neuron activation by harmaline involves attenuation of both HVA and LVA Ca(2+) conductances and consequent attenuation of Ca(2+)-sensitive K(+) conductances resulting in spike broadening and attenuation of the afterhyperpolarization. Both of HVA and LVA attenuation also suggests a role to regulate intracelluar Ca(2+), thereby to protect neurons from apoptosis.

Late‐onset bursts evoked by mossy fibre bundle stimulation in unipolar brush cells: evidence for the involvement of H‐ and TRP‐currents

Synaptic transmission at central synapses has usually short latency and graded amplitude, thereby regulating threshold crossing and the probability of action potential generation. In the granular layer of the vestibulo-cerebellum, unipolar brush cells (UBCs) receive a giant synapse generating a stereotyped excitatory postsynaptic potential (EPSP)-burst complex with early-onset (∼2 ms) and high reliability. By using patch-clamp recordings in cerebellar slices of the rat vestibulo-cerebellum, we found that mossy fibre bundle stimulation also evoked (in ∼80% of cases) a late-onset burst (after tens to hundreds of milliseconds) independent of EPSP generation. Different from the early-onset, the late-onset burst delay decreased and its duration increased by raising stimulation intensity or the number of impulses. Although depending on synaptic activity, the late-onset response was insensitive to perfusion of APV ((2R)-5-amino-phosphonopentanoate), NBQX (2,3-dioxo-6-nitro-tetrahydrobenzo(f)quinoxaline-7-sulfonamide) and MCPG ((RS)-α-methyl-4-carboxyphenylglycine) and did not therefore depend on conventional glutamatergic transmission mechanisms. The late-onset response was initiated by a slow depolarizing ramp driven by activation of an H-current (sensitive to ZD7288 and Cs(+)) and of a TRP- (transient receptor potential) current (sensitive to SKF96365), while the high voltage-activated and high voltage-activated Ca(2+) currents (sensitive to nimodipine and mibefradil, respectively) played a negligible role. The late-onset burst was occluded by intracellular cAMP. These results indicate that afferent activity can regulate H- and TRP-current gating in UBCs generating synaptically driven EPSP-independent responses, in which the delay rather than amplitude is graded with the intensity of the input pattern. This modality of synaptic transmission may play an important role in regulating UBC activation and granular layer functions in the vestibulo-cerebellum.

Bisphenol A Inhibits Voltage-Activated Ca2+ Channels in Vitro: Mechanisms and Structural Requirements

Bisphenol A (BPA), a high volume production chemical compound attracts growing attention as a health-relevant xenobiotic in humans. It can directly bind to hormone receptors, enzymes, and ion channels to become biologically active. In this study we show that BPA acts as a potent blocker of voltage-activated Ca(2+) channels. We determined the mechanisms of block and the structural elements of BPA essential for its action. Macroscopic Ba(2+) / Ca(2+) currents through native L-, N-, P/Q-, T-type Ca(2+) channels in rat endocrine GH(3) cells, mouse dorsal root ganglion neurons or cardiac myocytes, and recombinant human R-type Ca(2+) channels expressed in human embryonic kidney (HEK) 293 cells were rapidly and reversibly inhibited by BPA with similar potency (EC(50) values: 26-35 μM). Pharmacological and biophysical analysis of R-type Ca(2+) channels revealed that BPA interacts with the extracellular part of the channel protein. Its action does not require intracellular signaling pathways, is neither voltage- nor use-dependent, and does not affect channel gating. This indicates that BPA interacts with the channel in its resting state by directly binding to an external site outside the pore-forming region. Structure-effect analyses of various phenolic and bisphenolic compounds revealed that 1) a double-alkylated (R-C(CH(3))(2)-R, R-C(CH(3))(CH(2)CH(3))-R), or double-trifluoromethylated sp(3)-hybridized carbon atom between the two aromatic rings and 2) the two aromatic moieties in angulated orientation are optimal for BPA's effectiveness. Since BPA highly pollutes the environment and is incorporated into the human organism, our data may provide a basis for future studies relevant for human health and development.

Wide propagation of graded signals in nonspiking neurons

Signal processing in neuritic trees is ruled by the concerted action of passive and active membrane properties that, together, determine the degree of electrical compartmentalization of these trees. We analyzed how active properties modulate spatial propagation of graded signals in a pair of nonspiking (NS) neurons of the leech. NS neurons present a very extensive neuritic tree that mediates the interaction with all the excitatory motoneurons in leech ganglia. NS cells express voltage-activated Ca(2+) conductances (VACCs) that, under certain experimental conditions, evoke low-threshold spikes. We studied the distribution of calcium transients in NS neurons loaded with fluorescent calcium probes in response to low-threshold spikes, electrical depolarizing pulses, and synaptic inputs. The three types of stimuli evoked calcium transients of similar characteristics in the four main branches of the neuron. The magnitude of the calcium transients evoked by electrical pulses was a graded function of the change in NS membrane potential and depended on the baseline potential level. The underlying VACCs were partially inactivated at rest and strongly inactivated at -20 mV. Stimulation of mechanosensory pressure cells evoked calcium transients in NS neurons whose amplitude was a linear function of the amplitude of the postsynaptic response. The results evidenced that VACCs aid an efficient propagation of graded signals, turning the vast neuritic tree of NS cells into an electrically compact structure.