Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

Analysis of calcium channels in single spines using optical fluctuation analysis

Most synapses form on small, specialized postsynaptic structures known as dendritic spines. The influx of Ca2+ ions into such spines--through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)--triggers diverse processes that underlie synaptic plasticity. Using two-photon laser scanning microscopy, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1-20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (approximately 0.5) following dendritic action potentials. Activation of GABA(B) receptors reduced this probability in apical spines to approximately 0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.     

Dendritic spikes as a mechanism for cooperative long-term potentiation

Strengthening of synaptic connections following coincident pre- and postsynaptic activity was proposed by Hebb as a cellular mechanism for learning. Contemporary models assume that multiple synapses must act cooperatively to induce the postsynaptic activity required for hebbian synaptic plasticity. One mechanism for the implementation of this cooperation is action potential firing, which begins in the axon, but which can influence synaptic potentiation following active backpropagation into dendrites. Backpropagation is limited, however, and action potentials often fail to invade the most distal dendrites. Here we show that long-term potentiation of synapses on the distal dendrites of hippocampal CA1 pyramidal neurons does require cooperative synaptic inputs, but does not require axonal action potential firing and backpropagation. Rather, locally generated and spatially restricted regenerative potentials (dendritic spikes) contribute to the postsynaptic depolarization and calcium entry necessary to trigger potentiation of distal synapses. We find that this mechanism can also function at proximal synapses, suggesting that dendritic spikes participate generally in a form of synaptic potentiation that does not require postsynaptic action potential firing in the axon.     

Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.

Calcium has an important role in regulating epithelial cell ion transport and is itself transported by tissues involved in the maintenance of extracellular Ca2+ homeostasis. Although the mechanism of Ca2+ entry in electrically excitable cells is well-documented little is known about it in epithelial cells. Calcium absorption in polarized epithelial cells is a two-step process in which Ca2+ enters cells across apical plasma membranes and is extruded across basolateral membranes. Efflux may be mediated by an energy-dependent Ca2(+)-ATPase or by Na+/Ca2+ exchange. We examined Ca2+ influx in single cultured cells from distal renal tubules sensitive to parathyroid hormone by measuring intracellular Ca2+. Our results demonstrate that parathyroid hormone activates dihydropyridine-sensitive channels responsible for Ca2+ entry. We also show that microtubule-dependent exocytosis stimulated by parathyroid hormone may be necessary for the insertion or activation of Ca2+ channels in these cells. Once inserted or activated, dihydropyridine-sensitive channels mediate Ca2+ entry into these Ca2(+)-transporting epithelial cells. Our results support the view that agonist-induced exocytosis may represent a general paradigm for modulation of transport in epithelial cells by delivery and incorporation of transport proteins to plasma membranes or by delivery to plasma membranes of factors regulating these proteins.     

NMDA spikes in basal dendrites of cortical pyramidal neurons

Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 +/- 19.8 ms (half-width) cable-filtered voltage change at the soma that amplified the somatic voltage response by 226 +/- 46%. These spikes were accompanied by large calcium transients restricted to the activated dendritic segment. In contrast to conventional sodium or calcium spikes, these spikes were mediated mostly by NMDA (N-methyl-D-aspartate) receptor channels, which contributed at least 80% of the total charge. The ionic mechanism of these NMDA spikes may allow 'dynamic spike-initiation zones', set by the spatial distribution of glutamate pre-bound to NMDA receptors, which in turn would depend on recent and ongoing activity in the cortical network. In addition, NMDA spikes may serve as a powerful mechanism for modification of the cortical network by inducing long-term strengthening of co-activated neighbouring inputs.     

Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.     

Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites

Information in neurons flows from synapses, through the dendrites and cell body (soma), and, finally, along the axon as spikes of electrical activity that will ultimately release neurotransmitters from the nerve terminals. However, the dendrites of many neurons also have a secretory role, transmitting information back to afferent nerve terminals. In some central nervous system neurons, spikes that originate at the soma can travel along dendrites as well as axons, and may thus elicit secretion from both compartments. Here, we show that in hypothalamic oxytocin neurons, agents that mobilize intracellular Ca(2+) induce oxytocin release from dendrites without increasing the electrical activity of the cell body, and without inducing secretion from the nerve terminals. Conversely, electrical activity in the cell bodies can cause the secretion of oxytocin from nerve terminals with little or no release from the dendrites. Finally, mobilization of intracellular Ca(2+) can also prime the releasable pool of oxytocin in the dendrites. This priming action makes dendritic oxytocin available for release in response to subsequent spike activity. Priming persists for a prolonged period, changing the nature of interactions between oxytocin neurons and their neighbours.     

A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.     

Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger

Na+/Ca2+ exchange is electrogenic and moves one net positive charge per cycle. Although the cardiac exchanger has a three-to-one Na+/Ca2+ stoichiometry, details of the reaction cycle are not well defined. Here we associate Na+ translocation by the cardiac exchanger with positive charge movement in giant membrane patches from cardiac myocytes and oocytes expressing the cloned cardiac Na+/Ca2+ exchanger. The charge movements are initiated by step increments of the cytoplasmic Na+ concentration in the absence of Ca2+. Giant patches from control oocytes lack both steady-state Na+/Ca2+ exchange current (INaCa) and Na(+)-induced charge movements. Charge movements indicate about 400 exchangers per micron 2 in guinea-pig sarcolemma. Fully activated INaCa densities (20-30 microA cm-2) indicate maximum turnover rates of 5,000 s-1. As has been predicted for consecutive exchange models, the apparent ion affinities of steady state INaCa increase as the counterion concentrations are decreased. Consistent with an electroneutral Ca2+ translocation, we find that voltage dependence of INaCa in both directions is lost as Ca2+ concentration is decreased. The principal electrogenic step seems to be at the extracellular end of the Na+ translocation pathway.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.     

Ion permeation through the Na+,K+-ATPase

P-type ATPase pumps generate concentration gradients of cations across membranes in nearly all cells. They provide a polar transmembrane pathway, to which access is strictly controlled by coupled gates that are constrained to open alternately, thereby enabling thermodynamically uphill ion transport (for example, see ref. 1). Here we examine the ion pathway through the Na+,K+-ATPase, a representative P-type pump, after uncoupling its extra- and intracellular gates with the marine toxin palytoxin. We use small hydrophilic thiol-specific reagents as extracellular probes and we monitor their reactions, and the consequences, with cysteine residues introduced along the anticipated cation pathway through the pump. The distinct effects of differently charged reagents indicate that a wide outer vestibule penetrates deep into the Na+,K+-ATPase, where the pathway narrows and leads to a charge-selectivity filter. Acidic residues in this region, which are conserved to coordinate pumped ions, allow the approach of cations but exclude anions. Reversing the charge at just one of those positions converts the pathway from cation selective to anion selective. Close structural homology among the catalytic subunits of Ca2+-, Na+,K+- and H+,K+-ATPases argues that their extracytosolic cation exchange pathways all share these physical characteristics.     

Intracellular Ca indicator Quin-2 inhibits Ca2+ inflow via Na/Ca exchange in squid axon

Until recently, intracellular free calcium has been amenable to measurement and investigation only in cells large enough to permit either microinjection of a suitable Ca sensor such as a aequorin or arsenazo III or insertion of a Ca-sensitive microelectrode. This constraint on cell size was removed by the development of the fluorescent Ca2+ -sensitive dye Quin-2 and its acetoxymethyl ester, which can be introduced into a wide range of cell types. A major requirement of any intracellular Ca2+ indicator is that it should not disturb intracellular Ca2+ homeostasis and Quin-2 is generally considered to be satisfactory in this respect. We now report that injection of Quin-2 into squid (Loligo forbesi) axons can almost completely abolish one component of Ca2+ entry--intracellular Na+ (Nai)-dependent Ca2+ inflow, which occurs via Na/Ca exchange. Mixtures of Ca and Quin-2 that buffer an ionized Ca2+ at close to physiological concentrations also block Nai-dependent Ca2+ influx but these same mixtures fail to block the extracellular Na+ (Na0)-dependent extrusion of Ca2+, showing that Quin-2 acts specifically on Ca2+ inflow.     

Oxygen uptake occurs faster than sodium pumping in bee retina after a light flash

When neurones are active there is an entry of Na+, which must subsequently be pumped out, and an increase in their oxygen consumption rate (Qo2). The Na+ pump derives its energy from ATP, splitting it into ADP and Pi, and it has reasonably been proposed that the changes in concentrations of ATP, ADP and Pi lead to a stimulation of the O2 consumption by the mitochondria and hence to a restoration of the stock of ATP. Here we present evidence suggesting that Qo2 must be controlled differently in the retinal photoreceptor cells of the honeybee drone. Stimulation of drone photoreceptors with a flash of light causes an entry of Na+ (ref. 4) and a transient increase in Qo2 that indicates respiration of the right order of magnitude to provide ATP to pump the Na+ out. We report intracellular recordings of changes in intracellular sodium (Nai+) and potassium (Ki+) in response to single light flashes and have compared the time course of extra oxygen consumption (delta Qo2) with these ion changes and other indices of Na+ pumping. We found that the time course of pumping seems to lag behind the time course of delta Qo2. It follows that the mitochondrial respiration must be stimulated by some signal which is generated earlier than the rise in ADP produced by the Na+ pump.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Possible association of alcohol tolerance with increased synaptic Ca2+ sensitivity

The inhibitory effect of ethanol on neurotransmitter release has been suggested to be due to either reduced Ca2+ entry or increased removal of free intracellular Ca2+ from the synapse. The use of the Ca2+ ionophore, A23187, to allow direct access of external Ca2+ to the presynaptic interior should help to determine which of these two factors is the more important, as ethanol should inhibit A23187-induced release of transmitter only if increased Ca2+ removal from the synapse is important. Here we show in rat striatal slices that, although 3H-dopamine release evoked by depolarization with 40 mM K+ is inhibited by 50 mM ethanol, the release evoked by A23187 is enhanced by the presence of ethanol in vitro. The results suggest that ethanol reduces depolarization-induced transmitter release by reducing Ca2+ entry to the presynaptic terminal. However, for brain slices taken from rats made tolerant to ethanol, 3H-dopamine release in the absence of ethanol showed altered characteristics; both K+ depolarization and A23187 released a significantly greater fraction of 3H-dopamine from these slices than from controls. Thus tolerance to the inhibitory effect of ethanol on release may develop by a mechanism involving increased sensitivity of the terminal to Ca2+ entry.     

碱胁迫对宁夏枸杞叶中Na＋，K＋，Ca2＋动态变化的影响

摘要：为探讨宁夏枸杞叶中离子平衡与盐碱胁迫的关系,研究不同浓度的NaHCO3溶液胁迫下,枸杞叶中Na^＋,K^＋,Ca^2＋的浓度变化,同时采用非损伤微测技术研究了枸杞叶中Na^＋,K^＋,Ca^2＋的流速变化．结果表明,在同一时间内（7,14,21d）,Na^＋的浓度随NaHCO。浓度的升高总体呈升高趋势,K^＋和Ca^2＋的浓度总体呈下降趋势,c（Na^＋）／c（Ca^2＋）随NaHCO3浓度的升高而升高;随着时间的变化,各个处理下枸杞叶中Na^＋的浓度总体呈现先降后升的趋势,K^＋的浓度总体呈现下降趋势,Ca^2＋的浓度总体呈现先升后降的趋势,c（Na^＋）／c（K^＋）总体呈现升高趋势,c（Na^2＋）／c（Ca^2＋）总体呈现先降后升的趋势;NaHCO。溶液胁迫7d时,诱导了枸杞叶肉细胞中净Na^＋,K^＋,Ca^2＋外排的增加．碱胁迫下造成c（Na^＋）／c（K^＋）和f（Na^＋）／c（Ca^2＋）升高的原因为,叶片中K^＋和Ca^2＋外排和Na^＋大量积累,这也是枸杞不耐碱的原因之一．可为种植枸杞改良盐碱地提供参考．     

Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells

Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.