Chapter

A Lived History of Early Calcium Channel Discoveries Over the Past Half-Century

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Abstract

This chapter of the book is directed to PhD students, post-docs and young researchers who are attracted by the unique properties of voltage-gated calcium channels. The chapter aims to provide an overview of the most important discoveries that helped elucidate the structure and function of calcium channels in excitable cells. While systematically reviewing the numerous works in the field, I chose to write a personal story derived from my own experience on Ca2+ channels, as it developed in the lab and through the discussions with many colleagues working on ion channels. This occurred in a period in which Ca2+ channels reached maximal attention among scientists and brought the many astonishing achievements described in this book. Given the broad interdisciplinarity of Ca2+ channel discoveries, the present history may probably appear incomplete, but certainly, the other chapters of the book will cover all possible gaps on the matter.

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Ca(2+) plays a key role in intracellular signaling and controls various cellular processes such as proliferation, differentiation, cell growth, death, and apoptosis. Aberrant changes in intracellular Ca(2+) levels can promote undesired cell proliferation and migration and are therefore associated with certain tumor types. Many research groups have suggested a potential role for voltage-gated Ca(2+) channels in the regulation of tumor growth and progression, particularly T-type channels due to their unique biophysical properties. T-type channels are expressed in normal tissues throughout the body and in different types of tumors such as breast carcinoma, retinoblastoma, neuroblastoma, and glioma. It has been demonstrated that increased functional expression of the α1 subunit of T-type channels plays a role in the abnormal proliferation of glioblastoma cells. As such, siRNA-mediated knockdown of the expression of the α1 subunit of T-type channels decreases the proliferation of these cells. Moreover, pharmacological blockade of T-type channels significantly decreases tumor growth. In this review, we focus on the use of T-type channel blockers for the potential treatment of cancers, particularly highly proliferative tumors such as glioblastoma. We conclude that T-type channel blockers such as endostatin can serve as a potential therapeutic tool for tumors whose proliferation depends on increased T-type channel expression.
Article
ABSTRACT The calcium,current,of bullfrog,sympathetic,neurons,activates and deactivates rapidly (7 < 3 ms). For brief depolarizations, the current can be fit rea- sonably,well by a Hodgkin-Huxley-t,ype model,with,a single gating,particle of charge + 3. With 2 mM Ca 2+ as the charge carrier, half-maximal activation occurs at ~- 5 mV, near the voltage where activation and deactivation are slowest. When extracellular divalent ion concentrations are reduced, monovalent ions (e.g., Na § and methylammonium),produce kinetically similar inward currents. Current car- ried by Ba ~+ is blocked by Cd ~+ at micromolar concentrations, and by 100 nM w-conotoxin. Commercially available saxitoxin blocks the current, but different batches,have,quantitatively,different potency. The dihydropyridine,agonist Bay K 8644 induces a slight shift in activation kinetics to more negative voltages, with little effect on the peak,current. Nifedlpine at least partially reverses the effect of Bay K 8644, but has little effect on its own. Muscarinic agonists and other ligands that inhibit the M-type potassium,current,of frog sympathetic,neurons,have,weak inhibitory,effects on,the calcium,current,as well. One,interpretation,of these results is that the N-type calcium current predominates in these cells, with a minor contribution,of L-type current.
Article
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol.116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).
Article
Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.
Article
RELEASE of neurotransmitters from presynaptic axon terminals requires the influx of Ca2+ ions during the nerve terminal action potential1. Action potentials recorded in some neurone cell bodies exhibit a relatively large Ca2+ component, and it has been suggested that these soma Ca2+ spikes may provide a model for Ca2+ influx across the less accessible nerve terminal membrane2. Recent data support the usefulness of this model. Serotonin (5-hydroxytryptamine, 5-HT) increases transmitter output at certain habituated sensory nerve-motoneurone synapses in the abdominal ganglion of Aplysia and it also prolongs the Ca2+ spike recorded in the sensory neurone cell body3. Enkephalin reduces the stimulated release of substance P by adult cat trigeminal neurones4 and by cultured embryonic chick dorsal root ganglion (DRG) neurones5, and it decreases the quantal content of excitatory postsynaptic potentials (e.p.s.ps, transmitter unknown) evoked in cultured rat spinal cord neurones by co-cultured DRG cells6. This peptide also decreases the duration and magnitude of the Ca2+ component of the DRG soma spike5. With the thought that modulation of Ca2+ currents may be a general correlate of presynaptic inhibition, we have studied the effect of several putative neurotransmitters on the soma spike of cultured chick sensory neurones, and report here that they decrease the calcium component of cell body action potentials.
Article
Voltage gated calcium channels (Ca(2+) channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of Ca(V)2 (N- and P/Q-type) Ca(2+)-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of Ca(V)2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. This article is part of a Special Issue entitled: Calcium channels. http://dx.doi.org/10.1016/j.bbamem.2012.10.004
Article
High-voltage-activated (HVA) Ca-channel currents in chick sensory neurons were characterized by dihydropyridine compounds (DHPs) and -conotoxin GVIA (CTX) using patch-clamp methods. In single-channel recordings, two HVA-currents were identified by their single-channel conductances, 13 pS and 25 pS in 110 mM BaCl2. DHPs selectively affected the large-conductance channel. CTX (5 M), on the other hand, irreversibly eliminated only the small-conductance channel, while the large-conductance channel was either unaffected or only transiently blocked. In whole-cell recordings the macroscopic HVA-current was completely and irreversibly blocked by CTX but insensitive to DHPs in 60% of the cells. This current presumably was carried by the 13 pS channel. In the remaining cells, a part of the HVA-current (10%, SD=11%) was either unaffected or transiently blocked by CTX and was sensitive to DHPs. This current presumably was carried by the 25 pS channel. Inactivation of both macroscopic current component was incomplete during a 150 ms long depolarization. Our data suggest that the HVA-currents in chick sensory neurons are carried by two distinct Ca-channels that are differentially affected by CTX and DHPs.
Article
Ba currents flowing through single Ca-channels were recorded from cell-attached patches on rat pancreatic -cells. Two types of voltage-activated Ca-channels were found. The first (T-type) had a single channel conductance of 8 pS in 100 mM Ba, was activated at a low threshold (around 50mV) and inactivated by holding potentials positive to 40 mV. These properties are similar to those described for T-type channels in other preparations. The second type of Ca-channel (L-type) had a single channel conductance of 20pS in 100 mM Ba, was activated at a higher threshold (> 30mV), showed little inactivation during a 250 ms pulse and could be activated from a holding potential of 40mV. The dihydropyridine agonist BAYK 8644 selectively prolonged L-type Ca-channel openings. These properties are characteristic of L-type Ca-channels.
Article
Partially purified fractions of dihydropyridine and phenylalklyamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalklyamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.
Article
Voltage-gated calcium (Ca(2+)) channels are key transducers of membrane potential changes into intracellular Ca(2+) transients that initiate many physiological events. There are ten members of the voltage-gated Ca(2+) channel family in mammals, and they serve distinct roles in cellular signal transduction. The Ca(V)1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The Ca(V)2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The Ca(V)3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca(2+) channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.
Article
Blocking agents of high selectivity are crucial in defining both physiologically and biochemically the molecular components that control membrane excitability. To obtain such probes for voltage-dependent ion channels, we have examined the venom of several American scorpions for the presence of polypeptide neurotoxins having the required properties. We report here that using voltage-clamped giant axons of the squid Loligo vulgaris we have identified in the venom of the scorpion Centruroides noxius Hoffmann a polypeptide (fraction II-11) that specifically depresses the peak permeability of K+ channels without affecting their voltage-dependent open-close kinetics. The venom also contains a polypeptide toxin (fraction II-10) that specifically depresses Na+ peak permeability with only minor effects on the activation-inactivation kinetics. Furthermore, the physiological effects of the whole venom on the squid giant axon can be assigned quantitatively to the combined action of the two polypeptides.
Article
1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
Article
THE ionic channel associated with the acetylcholine (ACh) receptor at the neuromuscular junction of skeletal muscle fibres is probably the best described channel in biological membranes. Nevertheless, the properties of individual channels are still unknown, as previous studies were concerned with average population properties. Macroscopic conductance fluctuations occurring in the presence of ACh were analysed to provide estimates for single channel conductance and mean open times1-3. The values obtained, however, depended on assumptions about the shape of the elementary conductance contribution-for example, that the elementary contribution is a square pulse-like event2. Clearly, it would be of great interest to refine techniques of conductance measurement in order to resolve discrete changes in conductance which are expected to occur when single channels open or close. This has not been possible so far because of excessive extraneous background noise. We report on a more sensitive method of conductance measurement, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.
Article
Under depolarizing voltage clamp of Paramecium an inward calcium current developed and subsequently relaxed within 10 milliseconds. The relaxation was substantially slowed when most of the extracellular calcium was replaced by either strontium or barium. Evidence is presented that the relaxation is not accounted for by a drop in electromotive force acting on calcium, or by activation of a delayed potassium current. Relaxation of the current must, therefore, result from an inactivation of the calcium channel. This inactivation persisted after a pulse, as manifested by a reduced calcium current during subsequent depolarization. Inactivation was retarded by procedures that reduce net entry of calcium, and was independent of membrane potential. The calcium channel undergoes inactivation as a consequence of calcium entry during depolarization. In this respect, inactivation of the calcium channel departs qualitatively from the behavior described in the Hodgkin-Huxley model of the sodium channel.
Article
1. The unfertilized egg of the tunicate, Halocynthia roretzi, was intracellularly perfused with various solutions. 2. The perfusion apparatus consisted of lower and upper compartments which were connected by a small glass funnel. A denuded egg cell without chorion was dropped into the funnel and brought into close contact with the glass wall of the funnel. The membrane of the egg faced to the lower compartment was ruptured by a slight difference of hydrostatic pressure and the inside of the egg was perfused with the internal solution flowing through the lower compartment. The current across the upper membrane was analysed by voltage-clamp technique. 3. The egg cell in contact with 400 mM-Na external solution and perfused intracellularly with 400 mM-Na for 30 min showed a relatively low Na reversal potential, +6 mV, in comparison with +60 mV in the intact egg in standard artificial sea water. The exchange efficiency was monitored by observing the shift of Na reversal potential during perfusion with high Na internal perfusate. 4. The internal perfusate containing F- ions stabilized the egg membrane and kept the excitability for 1--2 hr during the intracellular perfusion. With the internal F- perfusate the intracellular cationic content was changed to 400 mM-Na, K, Rb or Cs (external solution of 400 mM-Na) and permeability ratios of the egg Na channel were estimated as PNa:PK:PRb:PCs=1.0:0.14:0.05:0.04. The internal F- perfusate abolished Ca current which was consistently observed in the intact egg, while the internal Cl- perfusate kept both Na and Ca current as in the intact egg. However with the internal Cl- perfusate the egg cell could not be kept in good condition more than 20-30 min. 6. The effects of intracellular free Ca ions upon the egg Na and Ca channels were analysed by using Ca ion-buffered internal Cl- and high Na perfusate. The results showed that internal Ca ions above 10(-6) reduced the Ca current and enhanced the Na current at the same time. In the range between 10(-5) and 10(-4) M the Ca current became half of the control obtained with zero free Ca perfusate while the Na conductance at the zero current level doubled. The internal Ca ions above one mM seemed to abolish the Ca current and to reduce the Na current as well. The reciprocal effect of intracellular Ca ions upon the egg Na and Ca channels was demonstrated in the concentration range from 10(-6) to 10(-3) M.