A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca²⁺ signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.     

Molecular Physiology and Pharmacology of the Calcium Channels

The activity of voltage-operated calcium channels is of primary importance for integration of the signals conducted along neuronal processes and transsynaptic transmission of messages carried by them to other neurons. The structure and function of various types of calcium channels has been the object of numerous investigations, including those carried out for many years in our Institute. This lecture briefly summarizes the main data in this field, with special attention towards recent possibilities of specific pharmacological intervention into the activity of different types of calcium channels, which is of the highest importance for effective treatment of various abnormalities in the functioning of the nervous system.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

Chiral aspects of drug action at ion channels: a commentary on the stereoselectivity of drug actions at voltage-gated ion channels with particular reference to verapamil actions at the Ca2+ channel.

Ion channels may be considered as pharmacological receptors possessing specific drug binding sites with defined structure-activity relationships. Accordingly drug binding to ion channels is stereoselective. Interpretation of this stereoselectivity may be complex because of the existence of differences in affinity and access to different channel states. Such state-dependent interactions may give rise to quantitative and qualitative differences in stereoselectivity. The implications of such differences are reviewed for drug action at Na+, K+ and Ca2+ channels. Detailed attention is paid to the actions of verapamil enantiomers in the cardiovascular system where activities differ in vascular and cardiac tissues because of state-dependent interactions and stereoselective first-oass metabolism.     

Properties of membranous phospholipase C from WRK1 cell: sensitivity to guanylnucleotides and bacterial toxins

As previously described, WRK₁ plasma membrane possesses a vasopressin-sensitive phospholipase C [G. Guillon et al., FEBS Lett. 196, 155–159]. In the present study, we examined the sensitivity of this enzyme to guanylnucleotides. GTPγS induced a time- and dose-dependent stimulation of Ins(1,4,5)P₃ and Ins(1,4)P₂ accumulation. No accumulation of InsP₁, Ins(1,3,4)P₃ or Ins(1,3,4,5)P₄ occured under similar conditions. Gpp(NH)p produced the same effect but was less potent. GTP and a nonhydrolyzable analogue of ATP, App(NH)p, were without effect. Calcium also stimulated the phospholipase C activity in a time- and dose-dependent manner. In the absence of calcium, the activity of GTPγS was considerably reduced. Physiological calcium concentrations (between 10⁻⁸ and 10⁻⁷M), allowed maximal GTPγS stimulation of phospholipase C activity. In this system, the presence of vasopressin alone did not generate inositol phosphate accumulation. However, this hormone: (i) reduced the lag-time observed during GTPγS stimulation, (ii) increased the sensitivity of phospholipase C to GTPγS, and (iii) did not modify the stimulation of phospholipase C induced by maximal doses of GTPγS. Unlike sodium fluoride, GTPγS elicited an irreversible activation of phospholipace C. Calcium, GTPγS and sodium fluoride stimulated the phospholipase C activity via mechanisms sharing a common step, since their maximal effects were not additive. Cholera toxin treatment, known to produce complete ADP-ribosylation of ‘s’ subunits, partially reduced the basal and the maximal GTPγS-mediated stimulation of phospholipase C activity as well as that caused by vasopressin. This inhibition was not mimicked by treatment with either forskolin or pertussi toxin.     

L-type voltage-gated calcium channels: understanding function through structure

L-type voltage-gated calcium channels (VGCCs) are multisubunit membrane proteins that regulate calcium influx into excitable cells. Within the last two years there have been four separate reports describing the structure of the skeletal muscle VGCC determined by electron microscopy and single particle analysis methods. There are some discrepancies between the structures, as well as reports for both monomeric and dimeric forms of the channel. This article considers each of the VGCC structures in terms of similarities and differences with an emphasis upon translation of data into a biological context.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure,photoaffinity labeling and endogenous protein kinase activity

Tritiated analogues of the Ca²⁺ channel blockers such as [³H] PN200-110, [³H] verapamil and [³H] diltiazem have been used to identify and isolate Ca²⁺ antagonist receptors. The Ca²⁺ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca²⁺ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.     

植物钙吸收、转运及代谢的生理和分子机制

钙是植物必需的营养元素。酸性砂质土壤中含钙较少,导致在其土壤上生长的作物容易缺钙。另外由于果树果实、果菜类和包心叶菜类的蒸腾作用弱,导致果树和蔬菜普遍生理缺钙。根系维管束组织可能通过共质体和质外体两种途径进行钙素吸收,而果实则可通过非维管束组织直接吸收钙素。Ca2 通过Ca2 通道内流进入胞质,并通过Ca2 -ATPase和Ca2 /H 反向转运蛋白外流以保持胞质内低Ca2 浓度。为了应对植物发育和环境胁迫信号,Ca2 由质膜、液泡膜和内质网膜的Ca2 通道内流进入胞质,导致胞质Ca2 浓度迅速增加,产生钙瞬变和钙振荡,传递到钙信号靶蛋白,如钙调素、钙依赖型蛋白激酶及钙调磷酸酶B类蛋白,引起特异的生理生化反应。本文综述了植物钙素吸收、转运以及代谢研究的最新进展,包括植物对钙的需求和作物缺钙的原因,根系维管束组织及果实钙素吸收机理,Ca2 跨膜运输特性,钙的信使作用以及钙信号靶蛋白等方面内容。     

Molecular Tuning of an EF-Hand-like Calcium Binding Loop : Contributions of the Coordinating Side Chain at Loop Position 3

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca²⁺ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca²⁺ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca²⁺ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca²⁺ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.     

Properties of membranous phospholipase C from WRK1 cell: Sensitivity to guanylnucleotides and bacterial toxins

As previously described, WRK₁ plasma membrane possesses a vasopressin-sensitive phospholipase C [G. Guillon et al., FEBS Lett. 196, 155–159]. In the present study, we examined the sensitivity of this enzyme to guanylnucleotides. GTPγS induced a time- and dose-dependent stimulation of Ins(1,4,5)P₃ and Ins(1,4)P₂ accumulation. No accumulation of InsP₁, Ins(1,3,4)P₃ or Ins(1,3,4,5)P₄ occured under similar conditions. Gpp(NH)p produced the same effect but was less potent. GTP and a nonhydrolyzable analogue of ATP, App(NH)p, were without effect. Calcium also stimulated the phospholipase C activity in a time- and dose-dependent manner. In the absence of calcium, the activity of GTPγS was considerably reduced. Physiological calcium concentrations (between 10⁻⁸ and 10⁻⁷M), allowed maximal GTPγS stimulation of phospholipase C activity. In this system, the presence of vasopressin alone did not generate inositol phosphate accumulation. However, this hormone: (i) reduced the lag-time observed during GTPγS stimulation, (ii) increased the sensitivity of phospholipase C to GTPγS, and (iii) did not modify the stimulation of phospholipase C induced by maximal doses of GTPγS. Unlike sodium fluoride, GTPγS elicited an irreversible activation of phospholipace C. Calcium, GTPγS and sodium fluoride stimulated the phospholipase C activity via mechanisms sharing a common step, since their maximal effects were not additive. Cholera toxin treatment, known to produce complete ADP-ribosylation of ‘αs’ subunits, partially reduced the basal and the maximal GTPγS-mediated stimulation of phospholipase C activity as well as that caused by vasopressin. This inhibition was not mimicked by treatment with either forskolin or pertussi toxin.     

Potassium, sodium, calcium and glutamate-gated channels: pore architecture and ligand action

In the last decade, the idea of common organization of certain ion channel families exhibiting diverse physiological and pharmacological properties has received strong experimental support. Transmembrane topologies and patterns of the pore-facing residues are conserved in P-loop channels that include high-selective cation channels and certain ligand-gated channels. X-ray structures of bacterial K+ channels, KcsA, MthK and KvAP, help to understand structure-function relationships of other P-loop channels. Data on binding sites and mechanisms of action of ligands of K+, Na+, Ca2+ and glutamate gated ion channels are considered in view of their possible structural similarity to the bacterial K+ channels. Emphasized are structural determinants of ligand-receptor interactions within the channels and mechanisms of state-dependent action of the ligands.     

The cGMP-dependent protein kinase-gene,protein, and function

Special issue dedicated to Dr. Paul Greengard     

Basolateral membrane K permselectivity and regulation in bullfrog cornea epithelium

Summary Ion channels permeable to barium and calcium were reconstituted from theAplysia nervous system into phospholipid bilayers formed on the tips of patch electrodes. With asymmetrical concentrations of barium or calcium on the two sides of the bilayer, the single-channel currents reversed at the calculated barium or calcium reversal potentials, indicating that the channels were cation selective. Channels with conductances of 10, 25 and 36 pS were routinely observed. Calcium and barium were equally effective as charge carriers for the 36-pS channel, whereas magnesium was at least fifteenfold less effective. The gating of all three channels was independent of the voltage across the bilayer, but was affected by the dihydropyridine calcium channel agonist Bay K 8644 (Bay K). In the presence of Bay K but not in its absence, long discrete gating events were routinely observed, suggesting that the dihydropyridine increased the probability of long open states as it does for calcium channels in other systems.Bilayers invariably contained more than a single channel (or conductance state). This was observed even when theAplysia nervous system membranes were prepared in the presence of cytoskeleton disrupting agents, or when the membrane proteins were diluted extensively with exogenous phospholipid. Furthermore, transitions between conductance levels were observed with high frequency. These findings, together with the fact that all of the conductance states share certain properties including voltage-independence and sensitivity to Bay K, suggest that the apparent multiple channel types may in fact represent subconductance states of a single ion channel.     

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.     

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na(+) current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3-S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

Calcium Uptake and Protein Phosphorylation in Myenteric Neurons, Like the Release of Vasoactive Intestinal Polypeptide and Acetylcholine, Are Frequency Dependent

The mechanism of the electrical-to-chemical decoding involved in the preferential release of the transmitters acetylcholine and vasoactive intestinal polypeptide (VIP) by electrical field stimulation at low (5 Hz) and high (50 Hz) frequencies was studied in superfused myenteric neurons. The stimulation-induced uptake of 45Ca2+ accompanying high frequency stimulation was markedly reduced by 10 microM nifedipine, a specific blocker of L-type voltage-sensitive Ca2+ channels (VSCCs), as was also the preferential high-frequency release of VIP. By contrast, the 45Ca2+ uptake during low-frequency stimulation was somewhat lower per pulse, and neither this uptake nor the preferential release of acetylcholine occurring at this frequency was significantly reduced by nifedipine. These findings suggest that the release of acetylcholine and VIP involve different VSCCs. The pattern of in vitro protein thiophosphorylation in tissue extracts of differentially stimulated myenteric neurons involved polypeptides of 205, 173, 86, 73, 57, 54, 46, 32, 28, and 24 kDa and was also markedly stimulus and nifedipine dependent. This suggests that different phosphoproteins are involved during the frequency-dependent activation of the different Ca2+ channels and exocytotic mechanisms.