Expression Of Voltage-dependent Calcium Channels Research Articles

Single-Vesicle Electrochemistry Reveals Sex Difference in Vesicular Storage and Release of Catecholamine.

Quantitative measurements of sex difference in vesicle chemistry (i.e., chemical storage and release) at the single-vesicle level are essential to understand sex differences in cognitive behaviors; however, such measurements are very challenging to conventional analytical methods. By using single-vesicle electrochemistry, we find the duration of single exocytotic events of chromaffin cells prepared from male rats is statistically longer than that from female rats, leading to more neurotransmitter released in the male group. Further analysis reveals that a higher percentage of vesicles in the female group release part of the neurotransmitter, i.e., partial release, during exocytosis than that in male group. This sex dimorphism in neurotransmitter release in exocytosis might relate to the sex difference in the expression of voltage-dependent calcium channels and membrane lipid composition. Our finding offers the first experimental evidence that sex dimorphism even exists in vesicle chemistry, providing a brand new viewpoint for understanding the sex dimorphism in exocytosis.

Attenuated β-adrenergic response in calcium/calmodulin-dependent protein kinase IV-knockout mice.

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the β-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to β-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the β-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac β-adrenergic signal transduction.

Regulation of GAP43/calmodulin complex formation via calcineurin-dependent mechanism in differentiated PC12 cells with altered PMCA isoforms composition.

Several lines of evidence suggest the contribution of age-related decline in plasma membrane calcium pump (PMCA) to the onset of neurodegenerative diseases. From four PMCA isoforms, PMCA2, and PMCA3 respond to a rapid removal of Ca2+ and are expressed predominantly in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in differentiated PC12 cells accelerated cell differentiation, but increased apoptosis in PMCA2-deficient line. We also demonstrated that altered expression of voltage-dependent calcium channels correlated with their higher contribution to Ca2+ influx, which varied between PMCA-reduced lines. Here, we propose a mechanism unique for differentiated PC12 cells by which PMCA2 and PMCA3 regulate pGAP43/GAP43 ratio and the interaction between GAP43 and calmodulin (CaM). Although down-regulation of PMCA2 or PMCA3 altered the content of GAP43/pGAP43, of paramount importance for the regulatory mechanism is a disruption of isoform-specific inhibitory PMCA/calcineurin interaction. In result, higher endogenous calcineurin (CaN) activity leads to hypophosphorylation of GAP43 in PMCA2- or PMCA3-deficient lines and intensification of GAP43/CaM complex formation, thus potentially limiting the availability of free CaM. In overall, our results indicate that both “fast” PMCA isoforms could actively regulate the local CaN function and CaN-downstream processes. In connection with our previous observations, we also suggest a negative feedback of cooperative action of CaM, GAP43, and CaN on P/Q and L-type channels activity. PMCAs- and CaN-dependent mechanism presented here, may signify a protective action against calcium overload in neuronal cells during aging, as well a potential way for decreasing neuronal cells vulnerability to neurodegenerative insults.

Lower density of L-type and higher density of P/Q-type of calcium channels in chromaffin cells of hypertensive, compared with normotensive rats

Enhanced activity of the sympatho-adrenal axis and augmented circulating catecholamines has been implicated in the development of hypertension. Release of catecholamine from stimulated adrenal medulla chromaffin cells has been shown to be higher and longer in spontaneously hypertensive rats (SHRs), compared with normotensive Wistar rats (NWRs). Whether differences in the functional expression of voltage-dependent calcium channels (VDCCs) of the L-, N-, or P/Q subtypes may contribute to such distinct secretory behaviour, is unknown. We therefore approached here this study in voltage-clamped NWR and SHR chromaffin cells, using 10mM Ba2+ as charge carrier (IBa) and selective blockers of each channel type. We found that compared with NWR cells, SHR chromaffin cells exhibited the following differences: (1) 30% diminution of the IBa fraction carried by L channels; (2) a doubling of the IBa fraction carried by P/Q channels; (3) more visible current modulation by ATP that could be linked to a 10-fold higher mRNA levels for purinergic receptors of the P2Y2 subtype; and (3) a higher contribution of PQ channels to the transients of the cytosolic calcium concentrations ([Ca2+]c) generated by K+, compared with L channels. These results may contribute to the better understanding of the greater calcium signalling and exocytotic responses of SHR compared with NWR chromaffin cells, found in three previous reports from our laboratories.

Role of n‐type voltage‐dependent calcium channels in autoimmune optic neuritis

The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker. We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.

NON‐L‐TYPE VOLTAGE‐DEPENDENT CALCIUM CHANNELS CONTROL VASCULAR TONE OF THE RAT BASILAR ARTERY

1. Constriction of cerebral arteries is considered to depend on L-type voltage-dependent calcium channels (VDCCs); however, many previous studies have used antagonists with potential non-selective actions. Our aim was to determine the expression and function of VDCCs in the rat basilar artery. 2. The relative expression of VDCC subtypes was assessed using quantitative polymerase chain reaction and immunohistochemistry. Data were correlated with physiological studies of vascular function. Domains I-II of the T channel subtypes expressed in the rat basilar artery were cloned and sequenced. 3. Blockade of L-type channels with nifedipine had no effect on vascular tone. In contrast, in the presence of nifedipine, hyperpolarization of short arterial segments produced relaxation, whereas depolarization of quiescent segments evoked constriction. 4. The mRNA and protein for L- and T-type VDCCs were strongly expressed in the main basilar artery and side branches, with Ca(V)3.1 and Ca(V)1.2 the predominant subtypes. 5. T-Type VDCC blockers (i.e. 1 micromol/L mibefradil, 10 micromol/L pimozide and 100 micromol/L flunarizine) decreased intracellular calcium in smooth muscle cells, relaxed and hyperpolarized arteries, whereas nickel chloride (100 micromol/L) had no effect. In contrast with nifedipine, 10 micromol/L nimodipine produced hyperpolarization and relaxation. 6. When arteries were relaxed with 10 micromol/L U73122 (a phospholipase C inhibitor) in the presence of nifedipine, 40 mmol/L KCl evoked depolarization and constriction, which was significantly reduced by 1 micromol/L mibefradil. 7. Sequencing of domains I-II revealed splice variants of Ca(V)3.1, which may impact on channel activity. 8. We conclude that vascular tone of the rat basilar artery results from calcium influx through nifedipine-insensitive VDCCs with pharmacology consistent with Ca(V)3.1 T-type channels.

GK domain of β subunit is essential for the expression of voltage-dependent calcium channels

GK domain of β subunit is essential for the expression of voltage-dependent calcium channels

Expression of voltage-dependent calcium channels in the embryonic rat midbrain

The diversity of expression of high-voltage activated voltage-dependent calcium channels (VDCC) was investigated with whole-cell voltage-clamp recordings from dissociated embryonic rat ventral mesencephalic cells over a 7-day culture period. Cell phenotype was identified post-recording by fluorescent immunocytochemistry as tyrosine hydroxylase positive (TH+) or glutamic acid decarboxylase positive (GAD+). Both TH+ and GAD+ cells displayed high-threshold calcium (Ca 2+) currents activated by depolarisations positive to −60 mV. In both cell types, pharmacological dissection using selective VDCC inhibitors, ω-agatoxin IVA (Aga IVA), ω-conotoxin GVIA (GVIA) and nifedipine demonstrated the existence of P/Q-, N- and L-type VDCC, respectively. The remaining residual current could be blocked by cadmium. It was found that the contribution to the whole-cell current by the N-type channel was greater in TH+ cells than GAD+ cells at each time point examined, whilst the contribution to the whole-cell current by the L-type channel was greater in GAD+ cells than TH+ cells. However, over the 7-day culture period, the expression of VDCC types in both cell phenotypes changed in a similar fashion, with the contribution to the whole-cell current from the N-type current decreasing, and the contribution from the R-type current increasing. Our data could provide new insights into a range of neurodevelopmental mechanisms related to Ca 2+ homeostasis in developing mesencephalic neurons.

Developmental changes in low and high voltage-activated calcium currents in acutely isolated mouse vestibular neurons.

1. The development of low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents was studied in neurons acutely dissociated from mouse vestibular ganglia at embryonic stages (E)14, 15, 17 and birth using the whole-cell patch-clamp technique. 2. LVA current was present in almost all neurons tested at stages E14 to E17, although at birth this current was restricted to a few neurons. Two populations of neurons were characterized based on the amplitude of the LVA current. In the first population, LVA current densities decreased between E17 and birth by which time this current tended to disappear in most neurons. A second population of neurons with high density LVA current appeared at E17, and in this group the mean density increased during development. 3. Among HVA currents, the dihydropyridine-sensitive L-type current remained constant between E15 and birth. Over the same period, the density of N- and Q-type currents continuously increased as shown using omega-conotoxin-GVIA (N-type), and high concentrations of omega-agatoxin-IVA (Q-type). The P-type current, sensitive to low concentrations of omega-agatoxin-IVA, transiently increased between E15 and E17, and then both current density and its proportion of the global current decreased. 4. Our results reveal large modifications in the expression of voltage-dependent calcium channels during embryonic development of primary vestibular neurons. The changes in the expression of LVA current and the transient augmentation of P-type HVA current occur during a period characterized by massive neuronal growth and by the beginning of synaptogenesis. These results suggest a specific role of these currents in the ontogenesis of vestibular primary afferents.

Mechanisms of modulation of voltage-dependent calcium channels by G proteins.

Voltage-dependent calcium channels (VDCCs) are key components in the complex functioning of excitable cells. Because of this, their regulation is of paramount importance to the control of cellular activity. VDCCs consist of a pore-forming α1 subunit, which has four domains each containing six putative transmembrane segments. From purification studies, these are associated with an intracellular β subunit. They also co-purify with an α2 subunit, which is entirely extracellular, linked into the membrane by S-S bonding to a transmembrane δ subunit (Witcher et al. 1993; Liu, De Waard, Scott, Gurnett, Lennon & Campbell, 1996) (Fig. 1). A number of different α1 subunits have been cloned; α1C, D and S all form 1,4-dihydropyridine (DHP)-sensitive L-type calcium channels, whereas α1A, B and E form P/Q-, N- and possibly R- or T-type channels, respectively. There are several means by which these channels may be modulated, but for neuronal channels, particularly N and P/Q, a major mechanism involves inhibitory modulation via the activation of heterotrimeric G proteins by seven transmembrane (7TM) receptors (for review see Dolphin, 1995). The key features of this inhibition are that it is always partial and is typified by a slowing of the current activation kinetics, which is thought to be due to a time-dependent recovery from voltage-dependent inhibition (Bean, 1989). The voltage dependence is manifested by a shift to more depolarized potentials of the current activation-voltage relationship, and the loss of inhibition at large depolarizations (Bean, 1989). There may also be additional mechanisms that are not voltage dependent, manifested by an incomplete ability of a depolarizing prepulse to reverse the inhibition (Diverse-Pierluissi & Dunlap, 1993), and the continuing presence of inhibition at large depolarizations measured from the tail current amplitude. Figure 1 The VDCC oligomeric complex Calcium channels are present in many tissues, where they fulfil a number of different specialized roles. In neurons the distribution of the channels is non-uniform, with α1B and α1A being particularly concentrated at synaptic terminals (Westenbroek, Hell, Warner, Dubel, Snutch & Catterall, 1992; Westenbroek et al. 1995). G protein-mediated modulation of these channels has been shown to occur at presynaptic terminals (Toth, Bindokas, Bleakman, Colmers & Miller, 1993). Evidence suggests that this mechanism may be responsible for at least some of the presynaptic inhibition of synaptic transmission mediated by a wide variety of 7TM receptors in many areas of the nervous system (Man-Son-Hing, Zoran, Lukowiak & Haydon, 1989; Hille, 1992; Toth et al. 1993; Dolphin, 1995). Activation of such receptors will reduce calcium entry into presynaptic terminals via VDCCs, but the effect should be frequency dependent. Inhibition will be reduced during a high frequency train as a result of the voltage dependence of the inhibitory modulation, providing a gain-setting mechanism. Relief of inhibition of calcium currents, evoked by action potential-like voltage waveforms, has been reported during high frequency trains (Williams, Serafin, Muhlethaler & Bernheim, 1997), and might contribute to the modulation of presynaptic inhibition depending on input frequency. The search for the molecular mechanism of this modulation is hotting up. Nevertheless, it should not be forgotten that indirect mechanisms such as presynaptic hyperpolarization, or direct mechanisms involving inhibition of exocytosis, are also likely to play a role in the modulation of presynaptic release of transmitter (Man-Son-Hing et al. 1989).

Immunohistochemical and in situ mRNA hybridisation techniques to determine the distribution of ion channels in human brain: a study of neuronal voltage-dependent calcium channels

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues [15]. Presynaptic neuronal VDCCs control neurotransmitter release [11, 8]and are important new drug targets [3]. They are composed of three subunits, α 1, β and α 2/ δ [4]and multiple gene classes of each protein have been identified [7, 12, 14]. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit [6, 13, 16].

S2-2 Voltage-dependent calcium channel expression and modulation by g proteins

S2-2 Voltage-dependent calcium channel expression and modulation by g proteins

S2-2 Voltage-dependent calcium channel expression and modulation by G proteins

S2-2 Voltage-dependent calcium channel expression and modulation by G proteins

Differentiated expression inXenopus oocytes of calcium channels from rat cerebellar and forebrain neurons

Calcium channels were expressed inXenopus laevis oocytes by means of matrix RNA (mRNA) extracted from the cerebellum (RNAc) and forebrain (RNAfb). In these oocytes, inward barium currents,I Ba, evoked by 40 mM Ba2+ were investigated using a double microelectrode technique. Currents expressed after injection of both RNAc and RNAfb (further referred to as RNAc- and RNAfb-expressed currents) showed a voltage-dependent characteristic typical of high-threshold calcium channels of mammalian neurons. The threshold of activation was about −40 mV, the maximum amplitude was observed at +20 mV and reversal potential at +60 mV. In both groups of oocytes, no expression of low- or high-threshold calcium channels of other types was observed. Although in both cases the expression ofI Ba had similar macrokinetics, characteristics of their stationary inactivation differed. The half-inactivation potential ranged between −32 and −16 mV, and the slope factor was 28 and 16.6 mV in RNAfb- and RNAc-injected oocytes, respectively. In both cases,I Ba were insensitive to dihydropyridines; however their relation to other pharmacological agents was different. RNAfb-expressedI Ba was completely blocked by Cd2+ (K d=10 µM) and depressed up to 70% by ω-conotoxin (1 µM), being insensitive to either whole spider toxin fromAgelenopsis aperta venom or to its FTX fraction. On the contrary, RNAc-expressedI Ba was more sensitive to Cd2+ (K d=0.1 µM), stable to ω-conotoxin, and suppressed up to 75–90% by wholeA. aperta toxin in a dilution of 1:10000, and by FTX at a concentration of 0.5 µM. The findings allow us to suggest that the forebrain and cerebellum of mammals are the structures, whose mRNA differ and provide predominant expression of voltage-dependent calcium channels of N- and P-types, respectively.

Differential depolarization-activated calcium responses in fetal and neonatal rat osteoblast-like cells

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 μM D600 or 10 μM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca 2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.