Interactions of divalent cations with single calcium channels from rat brain synaptosomes

Voltage-dependent calcium channels from a rat brain membrane preparation ("synaptosomes") were incorporated into planar lipid bilayers. The effects of calcium, barium, strontium, manganese, and cadmium ions on the amplitudes and kinetics of single channel currents were examined. The order of single channel conductances was gBa greater than gSr greater than gMn, which was the inverse of the order of the mean channel open times: TMn greater than TCa = TSr greater than TBa. In contrast, the identity of the charge carrier had little or no effect on the mean closed times of the channel. Manganese, in the absence of other permeant ions, can pass through single channels (gMn = 4 pS). However, when added to a solution that contained another type of permeant divalent cation, manganese reduced the single channel current in a voltage-dependent manner. Cadmium, a potent blocker of macroscopic "ensemble" calcium currents in many preparations, reduced the current through an open channel in a manner consistent with Cd ions both not being measurably permeant and interacting with a single site. The permeant ions competed with cadmium for this site with the following order: Mn greater than Sr = Ca greater than Ba. These results are consistent with the existence of no less than one divalent cation binding site in the channel that regulates ion permeation.     

Model of calcium channel inactivation: a qualitative analysis

A simple model of calcium channel inactivation has been developed, based on the accumulation of calcium ions at the inner mouth of the channel and on their binding to a receptor which inactivates the channel. A qualitative analysis has shown that upon an appropriate choice of parameters corresponding to the cell structure and to kinetic properties of its components, the calcium dependent inactivation and that assumed to be voltage dependent can both be emulated. The model suggests that the supposed variety of calcium channels might be explained by quantitative differences in nonlinear interactions of the channels with other cell components.     

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.     

The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of Caenorhabditis elegans consists of a syncytium of radially orientated muscle cells that contract synchronously and rhythmically to ingest and crush bacteria and pump them into the intestine of the animal. The action potentials that support this activity are superficially similar to vertebrate cardiac action potentials in appearance with a long, calcium-dependent plateau phase. Although the pharyngeal muscle can generate action potentials in the absence of external calcium ions, action potentials are absent when sodium is removed from the extracellullar solution (Franks et al. 2002). Here we have used whole cell patch clamp recordings from the pharynx and show low voltage-activated inward currents that are present in zero external calcium and reduced in zero external sodium ions. Whilst the lack of effect of zero calcium when sodium ions are present is not surprising in view of the known permeability of voltage-gated calcium channels to sodium ions, the reduction in current in zero sodium when calcium ions are present is harder to explain in terms of a conventional voltage-gated calcium channel. Inward currents were also recorded from egl-19 (n582) which has a loss of function mutation in the pharyngeal L-type calcium channel and these were also markedly reduced in zero external sodium. Despite this apparent dependence on external sodium ions, the current was partially blocked by the divalent cations, cadmium, barium and nickel. Using single-channel recordings we identified a cation channel for which the open-time duration was increased by depolarisation. In inside-out patches, the single-channel conductance was highest in symmetrical sodium solution. Further studies are required to determine the contribution of these channels to the pharyngeal action potential.     

Gating deficiency in a familial hemiplegic migraine type 1 mutant P/Q-type calcium channel

Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding alpha1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM alpha1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM alpha1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Ascorbic acid modulation of calcium channels in pancreatic beta cells

We have studied the effect of ascorbic acid on voltage-dependent calcium channels in pancreatic beta cells. Using the whole-cell and perforated-patch variants of the patch clamp technique to record calcium tail currents, we have shown that the slowly deactivating (SD) calcium channel, which is similar to the T-type channel in other cells, is inhibited in a voltage-dependent manner by ascorbic acid (AA). The other channels that carry inward current in beta cells, FD calcium channels and sodium channels, are unaffected by AA. Ascorbic acid causes a voltage-dependent decrease in the magnitude of the SD channel conductance which can be explained by the hypothesis that approximately 50-60% of the channels have their voltage dependence shifted by approximately 62 mV in the depolarizing direction. Thus, ascorbate appears to modify only a fraction of the SD channels. The activation kinetics of the ascorbate-modified channels are slower than control channels in a manner that is consistent with this hypothesis. Deactivation and inactivation kinetics are unaffected by ascorbate. These effects of ascorbate require metal ions, and it appears that some of the activity of ascorbate is due to a product of its metal catalyzed oxidation, perhaps dehydroascorbate.     

Dopamine modulates in a differential fashion T- and L-type calcium currents in bass retinal horizontal cells

White bass (Roccus chrysops) retinal horizontal cells possess two types of voltage-activated calcium currents which have recently been characterized with regard to their voltage dependence and pharmacology (Sullivan, J., and E. M. Lasater. 1992. Journal of General Physiology. 99:85-107). A low voltage-activated transient current was identified which resembles the T-type calcium current described in a number of other preparations, along with a sustained high threshold, long-lasting calcium current that resembles the L-type calcium current. Here we report on the modulation of horizontal cell calcium channels by dopamine. Under whole-cell voltage clamp conditions favoring the expression of both calcium currents, dopamine had opposing actions on the two types of voltage-sensitive calcium currents in the same cone- type horizontal cell. The L-type calcium current was significantly potentiated by dopamine while the T-type current was simultaneously reduced. Dopamine had no effect on calcium currents in rod-type horizontal cells. Both of dopamine's actions were mimicked with the D1 receptor agonist, SKF 38393, and blocked by application of the D1 specific antagonist, SCH 23390. Dopamine's actions on the two types of calcium currents in white bass horizontal cells are mimicked by the cell membrane-permeant cyclic AMP derivative, 8-(4-chlorophenylthio)- cyclic AMP, suggesting that dopamine's action is linked to a cAMP- mediated second messenger system. Furthermore, the inhibitor of cAMP- dependent protein kinase blocked both of dopamine's actions on the voltage-dependent calcium channels when introduced through the patch pipette. This indicates that protein phosphorylation is involved in modulating horizontal cell calcium channels by dopamine. Taken together, these results show that dopamine has differential effects on the voltage-dependent calcium currents in retinal horizontal cells. The modulation of these currents may play a role in shaping the response properties of horizontal cells.     

NALCN Ion Channels Have Alternative Selectivity Filters Resembling Calcium Channels or Sodium Channels

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG⁺ impermeant and blockable with 10 µM Gd³⁺ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.     

Mechanisms of permeation and selectivity in calcium channels

The mechanisms underlying ion transport and selectivity in calcium channels are examined using electrostatic calculations and Brownian dynamics simulations. We model the channel as a rigid structure with fixed charges in the walls, representing glutamate residues thought to be responsible for ion selectivity. Potential energy profiles obtained from multi-ion electrostatic calculations provide insights into ion permeation and many other observed features of L-type calcium channels. These qualitative explanations are confirmed by the results of Brownian dynamics simulations, which closely reproduce several experimental observations. These include the current-voltage curves, current-concentration relationship, block of monovalent currents by divalent ions, the anomalous mole fraction effect between sodium and calcium ions, attenuation of calcium current by external sodium ions, and the effects of mutating glutamate residues in the amino acid sequence.     

Disease-causing mutations of calcium channels

Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca2+ ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca2+ stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores. Mutations of genes encoding calcium have been implicated in the etiology of a diverse group of nerve and muscle diseases. These mutations have been identified in humans, mice and other organisms. In this review, we will summarize calcium channelopathies of humans and mice. Of the ten calcium channel α1 subunits cloned and sequenced (see ref. 1), disease-causing mutations have been found in CaV1.4 and CaV2.1 in the nervous system, and CaV1.1 and CaV1.2 in muscle. Mutations in calcium channel auxiliary subunits (α2δ, β and γ) have also been associated with both human and/or mouse neurological diseases. The disease-causing mutations may provide new insight into the cell biological roles of calcium channels as well as into relationships between structure and function. In addition, understanding how the mutations affect the physiology of the cell could lead to advances in disease treatment by relieving symptoms or slowing the progression of the disease. However, due to the multifaceted functions of calcium in the cell, the correlation between molecular mutation, physiological alterations and disease etiology is neither straightforward nor easily understood. Since calcium is an important intracellular signaling molecule, altered calcium channel function can give rise to widespread changes in cellular function. Indeed, serious diseases result from mutations that cause trivial alterations of calcium currents analyzed in vitro.     

Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes

Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.