Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

Modulatory effects of diadenosine polyphosphates on different types of calcium channels in the rat central neurons

Modulatory effects of diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A) on Ca²⁺ channels were studied on isolated hippocampal neurons and synaptosomes taken from the rat midbrain. In experiments on synaptosomes obtained from the whole brain, Ap₅A applied at a concentration of 100 µM increased the intrasynaptosomal calcium level (measured by means of spectrofluorometry) for 26±1.8 nM, i.e., by 24±2%. Nifedipine failed to block this effect in synaptosomes and in hippocampal neurons. The high voltage-activated Ca²⁺ currents were identified by recording from freshly isolatedCA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in control and after incubation with 5 µM Ap₅A. In the majority of tested pyramidal neurons, the latter procedure resulted in a reversible increase in the high voltage-activated currents through Ca²⁺ channels measured at a holding potential of –100 mV, but not of –40 mV. Potentiation of the currents through Ca²⁺ channels in hippocampal neurons as well as an increase in intrasynaptosomal [Ca²⁺] could be irreversibly blocked by 5 µM -conotoxin, but not by 200 nM -Aga-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type but not of L-type or P-type Ca²⁺ channels in many central neurons of the rat brain.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 409–416, November–December, 1994.     

Properties of two types of calcium channels in clonal pituitary cells

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.     

Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals

The properties of the Ca2+ channels mediating transmitter release in vertebrate neurons have not yet been described with voltage-clamp techniques. Several types of voltage-dependent Ca2+ channels are known to exist on neuronal somata, but the small size and inaccessibility of most vertebrate nerve endings have precluded direct characterization of the presynaptic channels. However, large nerve endings, which release the peptides oxytocin and vasopressin in a Ca2(+)-dependent manner, can be dissociated from the rat neurohypophysis. Using both single-channel and whole-cell patch-clamp techniques, we have characterized two types of Ca2+ channels that coexist in these terminals. One is a large-conductance, high-threshold, dihydropyridine-sensitive channel that contributes a slowly inactivating current. The second is a smaller conductance channel, which is also activated at high thresholds, but underlies a rapidly inactivating, dihydropyridine-insensitive current. Both types of Ca2+ channels may participate in the peptide release process.     

Specific functioning of Cav3.2 T-type calcium and TRPV1 channels under different types of STZ-diabetic neuropathy

Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8 weeks of diabetes development. Here we found that 6–7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception. The proportion of IB4-positive capsaicin-sensitive small DRG neurons, strongly involved in thermal nociception, was not altered under different types of PDN implying differential changes at cellular and molecular level. We further focused on properties of T-type calcium and TRPV1 channels, which are known to be involved in Ca^2 + signaling and pathological nociception. Indeed, TRPV1-mediated signaling in these neurons was downregulated under hypo- and normalgesia and upregulated under hyperalgesia. A complex interplay between diabetes-induced changes in functional expression of Cav3.2 T-type calcium channels and depolarizing shift of their steady-state inactivation resulted in upregulation of these channels under hyper- and normalgesia and their downregulation under hypoalgesia. As a result, T-type window current was increased by several times under hyperalgesia partially underlying the increased resting [Ca^2 +]_i observed in the hyperalgesic rats. At the same time Cav3.2-dependent Ca^2 + signaling was upregulated in all types of PDN. These findings indicate that alterations in functioning of Cav3.2 T-type and TRPV1 channels, specific for each type of PDN, may underlie the variety of pain syndromes induced by type 1 diabetes.     

Domains of calcium channels as dissipative structures in a simulated neuron

The lateral distribution of open calcium channels of the fluid-mosaic membrane were investigated in a phenomenological model of a cylinder-shaped nerve cell. The local density of the channels changed due to their lateral diffusion and voltage- and calcium-dependent conformation transitions between open and closed states. Domains with an increased steady density of the open calcium channels were created as a result of action of intracellular calcium on its own channels, increasing the probability of the open state of the latter. These spatially nonuniform distributions of the channels are considered dissipative structures emerged in the active nonlinear medium at the expense of energy of active transport.Neirofiziologiya/Neurophysiology, Vol. 26, No. 2, pp. 99–107, March–April, 1994.     

Regulation of calcium channels in brain: implications for the clinical neurosciences

Calcium is a major second messenger in neurons and modulates many neuronal functions, including protein phosphorylation, phospholipid metabolism, cytoskeletal activity, and neurotransmitter release. These important events, which regulate neuronal activity, are directly dependent on the influx of extracellular calcium through voltage-sensitive calcium channels (VSCCs) in the neuronal membrane. Modulation of VSCC function represents an important strategy for regulating neuronal excitability. Although substantial evidence supports the ability of dihydropyridines to block VSCCs and contractility in cardiovascular tissue, their ability to block the majority of neuronal VSCCs remains controversial. Benzodiazepines, and other anticonvulsants, block depolarization-dependent 45Ca uptake through VSCCs in brain synaptosome preparations. In addition, benzodiazepines reduce voltage-gated calcium conductance as determined by voltage clamp studies of identified invertebrate neurons. Inhibition of VSCC activity may be an important mechanism by which these compounds produce their anticonvulsant and sedative effects. Intrasomal injection of calcium-calmodulin-dependent protein kinase modulates calcium conductance in invertebrate neurons, suggesting that protein phosphorylation may be an endogenous regulatory mechanism of VSCC activity. Developing novel pharmacological approaches to regulating VSCCs and understanding the endogenous regulatory mechanisms may lead to new therapeutic approaches to the treatment of neurological diseases.     

Single-channel recordings of two types of calcium channels in rat pancreatic beta-cells.

Using the cell-attached configuration of the patch clamp technique, we have identified two different types of Ca channels in rat pancreatic beta-cell membranes. The two channels differ in single channel conductance, voltage dependence, and inactivation properties. The single-channel conductance, measured with 100 mM Ba2+ in the pipette, was 21.8 pS for the large channel and 6.4 pS for the small channel. The large-conductance channel is similar to the fast deactivating or L-type Ca channel described in other preparations. It is voltage dependent, has a threshold for activation around -30 mV, and can be activated from a holding potential of -40 mV. On the other hand, the small-conductance Ca channel is similar to the SD or T type Ca channel; it has a lower activation threshold, around -50 mV, and it can be inactivated by holding the membrane potential at -40 mV.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Mussel beds on different types of structures support different macroinvertebrate assemblages

Abstract Artificial structures, such as seawalls, pilings and pontoons, are common features of urban estuaries. They replace natural structures or add to the amount of hard substratum in an area and provide habitats for many fish and invertebrates. Previous work has concentrated on fish or on the invertebrates that occupy the primary substratum of artificial structures. Mussels often grow on different types of structures (pontoons, pilings, seawalls and natural reefs) and provide a secondary substratum for other organisms to inhabit. Counting and identifying organisms associated with mussel beds is traditionally done to species level, which is very time‐consuming. To save time, organisms in this study were identified to coarse levels of taxonomic resolution (a mix of taxa, such as class, order, family and genus), which showed similar patterns to those when particularly speciose and abundant groups were identified to species. This study tests hypotheses that the distribution and abundance of mobile and sessile organisms that inhabit mussel beds will differ among natural and various types of artificial structures. When the associated assemblages of mussel beds from different types of structures and from different locations were examined, assemblages varied according to the type of structure they inhabited and its location. Assemblages associated with mussels on pontoons differed consistently from those on other types of structures. Patterns in the assemblages were also consistent through time. These data show that the types and amounts of artificial structures added to an environment can affect the types, distribution and abundances of organisms living in biogenic habitats.     

The high affinity receptor for omega-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.     

中枢神经系统L-型电压门控钙通道的功能调控与脑缺血

中枢神经系统L 型电压门控钙通道 (L typevoltage gatedcalciumchannels ,L VGCCs)由α1C(D)亚基和辅助亚基组成。α1C亚基的C 端包含多个功能结构域 ,可分别与钙调素、钙调蛋白酶、cAMP依赖性蛋白激酶、Src家族酪氨酸蛋白激酶 (Srcfamilyproteintyrosinekinases ,SrcPTKs)等相互作用 ,从而参与L VGCCs的功能调控。SrcPTKs介导的两种钙通道———L VGCCs和N 甲基 D 天冬氨酸 (N methyl D aspartate ,NMDA)受体的对话可能是缺血性脑损伤的重要机制     

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.     

Rice two-pore K+ channels are expressed in different types of vacuoles

Potassium (K+) is a major nutrient for plant growth and development. Vacuolar K+ ion channels of the two-pore K+ (TPK) family play an important role in maintaining K+ homeostasis. Several TPK channels were previously shown to be expressed in the lytic vacuole (LV) tonoplast. Plants also contain smaller protein storage vacuoles (PSVs) that contain membrane transporters. However, the mechanisms that define how membrane proteins reach different vacuolar destinations are largely unknown. The Oryza sativa genome encodes two TPK isoforms (TPKa and TPKb) that have very similar sequences and are ubiquitously expressed. The electrophysiological properties of both TPKs were comparable, showing inward rectification and voltage independence. In spite of high levels of similarity in sequence and transport properties, the cellular localization of TPKa and TPKb channels was different, with TPKa localization predominantly at the large LV and TPKb primarily in smaller PSV-type compartments. Trafficking of TPKa was sensitive to brefeldin A, while that of TPKb was not. The use of TPKa:TPKb chimeras showed that C-terminal domains are crucial for the differential targeting of TPKa and TPKb. Site-directed mutagenesis of C-terminal residues that were different between TPKa and TPKb identified three amino acids that are important in determining ultimate vacuolar destination.     

The calcium rhythms of different cell types oscillate with different circadian phases

Transgenic tobacco (Nicotiana plumbaginifolia) seedlings containing the Ca(2+)-sensitive luminescent protein aequorin have been shown to exhibit circadian variations in cytosolic calcium. Concomitant measurements of cytosolic and nuclear calcium show that circadian variations in the cytoplasm are not expressed in the nucleus. To investigate whether all cells of transgenic seedlings contribute equally to circadian variations in cytosolic calcium, different promoters eliciting different expression patterns have been placed upstream of aequorin and used for transformation. The circadian peak occurred at different times in the three transgenic lines constructed. Luminescence imaging of these transgenic lines indicated that aequorin was differentially accumulated among the main tissues and cells of the seedlings and overcoat technology with applied epidermal strips indicated that the surface cell layers contribute the vast majority of luminescent light. We conclude that the Ca(2+) rhythmicities of cells and tissues oscillate with distinct differences in phase, that this might represent different underlying cellular control mechanisms and that these observations have significant implications for our understanding and study of Ca(2+) mediated signal transduction in plant cells.