Neuronal Ca Channels Research Articles

Characteristics of [125I]omega-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain.

Characteristic of [125I]omega-conotoxin (omega-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]:DSP) was systematically investigated in crude membranes from chick whole brain. [125I]omega-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [125I]omega-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [125I]omega-CgTX labeling of the 216 kDa band and specific [125I]omega-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [125I]omega-CgTX using DSP involves the specific binding sites of [125I]omega-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons.

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Characterization of single calcium channels in Drosophila embryonic nerve and muscle cells

Voltage-activated Ca channels play a central role in synaptic transmission, control of cell excitability, and many other cellular processes. It is now clear that there are multiple types of Ca channels with various modes of modulation. Drosophila offers exceptional advantages for studying the molecular basis of the diversity and modulation of Ca channels. As a step in this study, we have characterized the single-channel Ba currents recorded from cell-attached patches on cultured embryonic Drosophila nerve and muscle cells. The voltage dependence and selectivity of the channels carrying these Ba currents identify them as Ca channels. All Ca channels found in Drosophila neurons appear to have the same voltage dependence of activation and similar single-channel conductance, 12-17 pS (100 mM Ba2+). However, the kinetic properties of individual Ca channels vary greatly. The mean open time of individual channels ranges from 2 msec to less than 0.2 msec. Some channels completely inactivate during the first half of a 90 msec depolarization, while others are more active in the second half. Many channels open during almost every depolarization, while others open in less than 20% of the depolarizations. Channels with longer open times tend to inactivate and open during a small fraction of depolarizations. When these kinetic properties were quantified, a continuum of values was found, instead of the clustering of values that might be expected for discrete types of channels. Muscle Ca channels form a more homogeneous class than do the neuronal Ca channels. The muscle Ca channel conductance is 18 pS. These channels do not inactivate during 90 msec depolarizations and open during a majority of depolarizations applied. Muscle Ca channels are similar to a subset of neuronal Ca channels. When a purified toxin from the spider Hololena curta is applied to neurons, the number of active Ca channels is reduced, and those channels still active open in a small fraction of depolarization. Since channels that open in a small fraction of depolarizations tend to inactivate, these data support the hypothesis that this toxin selectively blocks noninactivating neuronal Ca channels. This differential toxin sensitivity and the much larger variability observed in kinetic properties of neuronal, compared to muscle, Ca channels suggest that there are at least two types of neuronal Ca channels in Drosophila.

Potential-dependent Ca Channels of neurons in the mollusc Lymnaea stagnalis in aging: Effect of norepinephrine

During aging changes occur in the function of potential-dependent Ca channels of plasmic membrane of identified neurons in the mollusc Lymnaea stagnalis, i.e. the value of Ca inward current of neurons in old vs. adult molluscs was much higher at holding potential − 50 – − 30 mV. When held at −20 mV, the amplitude of Ca current decreased in the neurons of old molluscs, but continued to increase in those of adult molluscs. Various populations of potential-dependent Ca channels are assumed to have irregular age-related changes. The norepinephrine-induced inhibition of Ca current was more significant in old vs. adult molluscan neurons.

Voltage-dependent inhibition and facilitation of Ca channel activation by GTP-γ-S and Ca-agonists in adult rat sensory neurons

Intracellular application of guanosine 5′- O-3-thiotriphosphate (GTP-γ-S, 100 μM) causes a slow down of high-threshold Ca channel activation in adult rat sensory neurons that is relieved by strong depolarizations or by the Ca agonist Bay K 8644 (5 μM). Recovery from GTP-γ-S inhibition is usually accompanied by an increase in Ca current amplitude (facilitation) and is insensitive to holding potential (−60 to −90 mV). Inhibition and facilitation of Ca currents are also little affected by nifedipine (5 μM) or by cell incubation with ω-conotoxin (ω-CgTx, 3.2 μM). We conclude that both slowdown of Ca channel activation by GTP-γ-S and facilitation by strong depolarizations or by Ca agonists derive from a common process in which G-protein activation, membrane voltage and Ca agonist receptors interact to modulate neuronal Ca channel gatings.

Ca currents in human neuroblastoma IMR32 cells: kinetics, permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The low-threshold (LVA, T) Ca current activated positive to -50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 microM omega-conotoxin (omega-CgTx) or 40 microM Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to -30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of 125I-omega-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 microM omega-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual omega-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 microM) had a clear agonistic action on omega-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the omega-CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T;L,N) may be widely misleading if not supported by pharmacological assays.

S-9-6 - Neuronal Ca channels in mammalian brain neuron

S-9-6 - Neuronal Ca channels in mammalian brain neuron

Action of Ca 2+ agonists/antagonists in mammalian peripheral neurons

The action of several ligands on the low- (LVA,T) and high-threshold (HVA,L and N) Ca channels of adult rat sensory neurons and human neuroblastoma IMR32 cells has been investigated. In both cell types, 40 μM Cd 2+ and 6.4 μM ¦ω-Conotoxin (ω-CgTx) selectively blocked the HVA channels, sparing the majority of LVA channels that were antagonized by amiloride and Ni 2+. In 50% of the cells, however, ¦ω-CgTx spared also a 15% of HVA channels that proved to be sensitive to BAY K 8644. The agonistic action of BAY K 8644 on ¦ω-CgTx-resistant HVA channels caused a large Ba current increase, prolonged current deactivation and acceleration of HVA channels inactivation that was particularly evident in adult rat DRG.

Solubilization, Partial Purification, and Properties of ω‐Conotoxin Receptors Associated with Voltage‐Dependent Calcium Channels from Rat Brain Synaptosomesa

These experiments provide a starting point for biochemical characterization of Ca channels from neuronal membranes, using omega-CgTX as a specific marker. The purification of the omega-CgTX receptors is far from complete. Each of the purification steps described results in only a two- to fivefold enrichment of the receptor proteins, and is accompanied by a loss of receptor concentration and stability, so the maximal specific activity achieved by a combination of these steps falls several orders of magnitude short of that of a large, homogeneous, active protein. Nevertheless, these studies have yielded important information about the omega-CgTX receptor. The Stokes' radius, determined from gel exclusion chromatography, is approximately 87 A, and the sedimentation coefficient, determined from sucrose gradient sedimentation, is approximately 19 S. These values are similar to those found for the DHP receptors solubilized in digitonin. We have also found that at least some of the omega-CgTX receptors have complex carbohydrate moieties that are recognized by WGA, together with evidence of heterogeneity of receptor glycosylation. Additionally, we have been able to use the solubilized, partially purified receptors in cross-linking experiments to tentatively identify the molecular weights of the omega-CgTX targets from rat brain. A large peptide of approximately 300 kDa, similar to that identified in photoaffinity studies, is very clearly labeled by the chemical incorporation of [125I]omega-CgTX into partially purified receptor preparations, but some ambiguity remains because of the faint labeling of peptides in the 120-170-kDa range. The approximately 300-kDa peptide is much larger than any single peptide component of DHP receptors from skeletal muscle, and it may be related to a molecular combination of the 170-kDa and 135-kDa subunits of the DHP receptor. Because [125I]omega-CgTX presumably labels both N- and L-type neuronal Ca channels, both channel types will probably be found in the purified preparations. Thus, at some time, it will be necessary to separate DHP-sensitive L-type channels from preparations of L- and N-type channels identified by omega-CgTX binding.

Omega-conotoxin blockade distinguishes Ca from Na permeable states in neuronal calcium channels.

The blocking properties of the neurotoxic peptide omega-conotoxin GVIA (omega-CgTX), on neuronal Ca channels were investigated. In line with previous reports (Feldman et al. 1987; McCleskey et al. 1987), we found that micromolar concentrations of the toxin block selectively and persistently the high-threshold Ca channels of chick sensory neurons. The block by omega-CgTX could be partially relieved in low [Ca2+]o (less than 1 microM) toxin-free solutions, allowing Na ions to flow through open high-threshold Ca channels. Ca currents through these channels, however, remained permanently blocked on returning to normal Ca2+ toxin-free solutions. Also neurons which were preincubated with omega-CgTX in low Ca2+ (5 mM EGTA) failed to show high-threshold Ca currents during washing with normal Ca2+. Thus, appearance of Na currents through Ca channels in CgTX-pretreated cells was neither a consequence of unbinding of the toxin from its receptor site nor due to an interaction of EGTA with bound omega-CgTX. Na currents in CgTX-pretreated cells could be reversibly suppressed by bath applications of verapamil or by further addition of the toxin. At variance with Ca currents, block of Na currents by omega-CgTX was faster and reversible (KD 0.7 microM). Our data are consistent with the idea that neuronal Ca channels are in different conformational states when permeable to Ca2+ or Na+ ions and that omega-CgTX depresses persistently ion permeation primarily in the Ca-permeable state.

Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using .omega.-conotoxin from Conus magus venom

The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.

Three types of neuronal calcium channel with different calcium agonist sensitivity

How many types of calcium channels exist in neurones? This question is fundamental to understanding how calcium entry contributes to diverse neuronal functions such as transmitter release, neurite extension, spike initiation and rhythmic firing. There is considerable evidence for the presence of more than one type of Ca conductance in neurones and other cells. However, little is known about single-channel properties of diverse neuronal Ca channels, or their responsiveness to dihydropyridines, compounds widely used as labels in Ca channel purification. Here we report evidence for the coexistence of three types of Ca channel in sensory neurones of the chick dorsal root ganglion. In addition to a large conductance channel that contributes long-lasting current at strong depolarizations (L), and a relatively tiny conductance that underlies a transient current activated at weak depolarizations (T), we find a third type of unitary activity (N) that is neither T nor L. N-type Ca channels require strongly negative potentials for complete removal of inactivation (unlike L) and strong depolarizations for activation (unlike T). The dihydropyridine Ca agonist Bay K 8644 strongly increases the opening probability of L-, but not T- or N-type channels.