Crayfish muscle: permeability to sodium induced by calcium depletion

Membrane of crayfish muscle fibers becomes selectively permeable to sodium when the calcium concentration of the bathing medium is reduced. Removal of calcium or its reduction below 1 or 2 millimole per liter causes large transient depolarizations up to 70 millivolts in amplitude. They resemble pro longed action potentials and occur only in the presence of sodium. The responses are abolished when tris(hydroxymethyl)aminomethane or lithium is substituted for sodium, and are blocked by tetrodotoxin even in the presence of sodium.     

Identification of an intracellular peptide segment involved in sodium channel inactivation

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.     

Two distinct populations of calcium channels in a clonal line of pituitary cells

The whole-cell variant of the patch clamp technique was used to study calcium channels in GH3 cells. Two distinct populations of calcium channels, first recognized from their closing kinetics, were observed. The slowly closing channels are activated in a relatively negative voltage range and are inactivated within 100 milliseconds. They conduct barium and calcium about equally well. The fast closing channels are activated at more positive voltages, are not inactivated during a 100-millisecond pulse, conduct barium in preference to calcium, and are activated slightly more rapidly than the slowly closing channels.     

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.     

Development of two types of calcium channels in cultured mammalian hippocampal neurons

Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.     

Ca2+ and Ca2+-activated K+ currents in mammalian gastric smooth muscle cells

Inward movement of calcium through voltage-dependent channels in muscle is thought to initiate the action potential and trigger contraction. Calcium-activated potassium channels carry large outward potassium currents that may be responsible for membrane repolarization. Calcium and calcium-activated potassium currents were identified in enzymatically isolated mammalian gastric myocytes. These currents were blocked by cadmium and nifedipine but were not substantially affected by diltiazem or D600. No evidence for a tetrodotoxin-sensitive sodium current or an inwardly rectifying potassium current was found.     

Functional differences between two classes of sodium channels in developing rat skeletal muscle

Excitability is generated in developing skeletal muscle by the incorporation of sodium-selective ion channels into the surface membrane. Whole-cell and patch voltage-clamp recording from myotubes and their embryologic precursors, myoblasts, indicated that voltage-activated sodium current in myoblasts was more resistant to block by tetrodotoxin (TTX) than that in myotubes. Single-channel recording from both cell types showed two classes of sodium channels. One class had a lower single-channel conductance, activated at more hyperpolarized voltages, and was more resistant to TTX than the other. The proportion of TTX-resistant to TTX-sensitive sodium channels was higher in myoblasts than in myotubes. Thus, the difference in TTX sensitivity between myoblasts and myotubes can be explained by a difference in the proportion of the two classes of sodium channels. In addition, the lower conductance of TTX-resistant channels provides insight into the relationship between the TTX binding site and the external mouth of the sodium channel.     

Atrionatriuretic peptide transforms cardiac sodium channels into calcium-conducting channels

The atrionatriuretic peptide (ANP) is released from atrial cells in response to increased extracellular fluid volume and reduces sodium absorption by the kidney, thus reducing the blood volume. In this report, ANP suppressed the calcium and sodium currents in rat and guinea pig ventricular myocytes. The suppression of sodium current was caused by enhanced permeability of the sodium channel to calcium without significant changes in the kinetics or the tetrodotoxin sensitivity of the channel. Thus, ANP may regulate the sodium channel by altering its cationic selectivity site to calcium, thereby repressing the sodium current. The suppression of sodium and calcium channels and the resultant depressed excitability of the atrial cells may help to regulate ANP secretion.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Permeation of calcium ions through non-NMDA glutamate channels in retinal bipolar cells.

The conduction of calcium ions through glutamate-gated channels is important in the induction of long-term potentiation and may trigger other cellular changes. In retinal bipolar cells, which lack the N-methyl-D-aspartate (NMDA) type of glutamate-gated channel, calcium permeability through non-NMDA channels was examined. Changes in extracellular calcium concentration unexpectedly affected the reversal potential for glutamate-induced currents in a manner consistent with these channels being highly permeable to calcium. External magnesium ions promote desensitization of these non-NMDA channels in a voltage-independent way. Thus, in addition to non-NMDA channels that conduct only sodium and potassium, there is a class that is also permeable to calcium.     

Atrotoxin: a specific agonist for calcium currents in heart

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.     

镉胁迫下NO对胡杨细胞Cd2+吸收调控机制的研究

本文研究了NO对胡杨愈伤细胞Cd2+耐受性的影响。结果表明:Cd2+(50 μmol/L)显著抑制了胡杨细胞的生长,而硝普钠SNP(NO供体,25 μmol/L)能明显缓解Cd2+对胡杨细胞生长的抑制作用,并减轻镉对细胞膜的伤害以及镉胁迫导致的细胞活力下降。利用非损伤微测技术等研究了NO对Cd2+动态吸收的影响。CdCl2(50 μmol/L)处理之后,胡杨细胞表现出Cd2+内流,而SNP(25 μmol/L,6 h)显著抑制了Cd2+的内流,并降低了Cd2+在细胞内的积累。研究发现,NO是通过调控钙离子通道来抑制胡杨细胞对Cd2+的吸收。镉胁迫下Cd2+内流被钙离子通道专一性抑制剂氯化镧明显抑制,表明Cd2+是通过钙离子通道转运进入细胞。并且发现,NO是通过促进Ca2+的内流来竞争性地抑制胡杨细胞对Cd2+的吸收,从而缓解了镉胁迫对胡杨细胞造成的生长抑制。      

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.     

Calmodulin activation of calcium-dependent sodium channels in excised membrane patches of Paramecium

Calmodulin is a calcium-binding protein that participates in the transduction of calcium signals. The electric phenotypes of calmodulin mutants of Paramecium have suggested that the protein may regulate some calcium-dependent ion channels. Calcium-dependent sodium single channels in excised patches of the plasma membrane from Paramecium were identified, and their activity was shown to decrease after brief exposure to submicromolar concentrations of calcium. Channel activity was restored to these inactivated patches by adding calmodulin that was isolated from Paramecium to the cytoplasmic surface. This restoration of channel activity did not require adenosine triphosphate and therefore, probably resulted from direct binding of calmodulin, either to the sodium channel itself or to a channel regulator that was associated with the patch membrane.     

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.     

Identification of an alpha subunit of dihydropyridine-sensitive brain calcium channels

Voltage-sensitive calcium channels in different tissues have diverse functional properties. Polyclonal antibodies (PAC-2) against the alpha subunits of purified rabbit skeletal muscle calcium channels immunoprecipitated calcium channels labeled with the dihydropyridine PN200-110 from both skeletal muscle and brain. The immunoreactivity of PAC-2 with the skeletal muscle channel was greater than that with the brain calcium channel and was absorbed only partially by prior treatment with the brain channel. PAC-2 specifically recognized a large peptide in synaptic plasma membranes of rabbit brain with an apparent molecular size of 169,000 daltons. This protein resembles an alpha subunit of the skeletal muscle calcium channel in apparent molecular weight, antigenic properties, and electrophoretic behavior after reduction of disulfide bonds. Thus, the dihydropyridine-sensitive calcium channel of rabbit brain has an alpha subunit that is homologous, but not identical, to those of the skeletal muscle calcium channel. The different functional properties of these two calcium channels may result from minor variations in structurally similar components.     

External calcium ions are required for potassium channel gating in squid neurons

The effects of calcium removal on the voltage-dependent potassium channels of isolated squid neurons were studied with whole cell patch-clamp techniques. When the calcium ion concentration was lowered from 10 to 0 millimolar (that is, no added calcium), potassium channel activity, identified from its characteristic time course, disappeared within a few seconds and there was a parallel increase in resting membrane conductance and in the holding current. The close temporal correlation of the changes in the three parameters suggests that potassium channels lose their ability to close in the absence of calcium and simultaneously lose their selectivity. If potassium channels were blocked by barium ion before calcium ion was removed, the increases in membrane conductance and holding current were delayed or prevented. Thus calcium is an essential cofactor in the gating of potassium channels in squid neurons.     

The molecular basis of acid insensitivity in the African naked mole-rat

Acid evokes pain by exciting nociceptors; the acid sensors are proton-gated ion channels that depolarize neurons. The naked mole-rat (Heterocephalus glaber) is exceptional in its acid insensitivity, but acid sensors (acid-sensing ion channels and the transient receptor potential vanilloid-1 ion channel) in naked mole-rat nociceptors are similar to those in other vertebrates. Acid inhibition of voltage-gated sodium currents is more profound in naked mole-rat nociceptors than in mouse nociceptors, however, which effectively prevents acid-induced action potential initiation. We describe a species-specific variant of the nociceptor sodium channel Na(V)1.7, which is potently blocked by protons and can account for acid insensitivity in this species. Thus, evolutionary pressure has selected for an Na(V)1.7 gene variant that tips the balance from proton-induced excitation to inhibition of action potential initiation to abolish acid nociception.     

Calcium channels in planar lipid bilayers: insights into mechanisms of ion permeation and gating

Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.     

Voltage-dependent calcium channels from Paramecium cilia incorporated into planar lipid bilayers

Two different divalent cation-selective channels from Paramecium cilia were incorporated into planar lipid bilayers. Both channels were much more permeable to divalent than univalent cations, and one of them discriminated significantly among the divalent cations. The selectivity and voltage dependence of the latter channel are comparable to those of voltage-dependent calcium channels found in a variety of cells. A combined biochemical, biophysical, and genetic study of calcium channels is now possible.