Channels In Excitable Cells Research Articles

CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation–transcription coupling

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation–transcription coupling we characterized the dependence of cAMP response element–binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca2+ channel pore has a remarkably mild effect, although some Ca2+ entry is absolutely required. Our data indicate that Ca2+/CaM-dependent protein kinase II acting near the channel couples local Ca2+ rises to signal transduction, encoding the frequency of Ca2+ channel openings rather than integrated Ca2+ flux—a form of digital logic.

A Mutation in Telethonin Alters Nav1.5 Function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.

Apical SK potassium channels and Ca2+‐dependent anion secretion in endometrial epithelial cells

Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (I(sc)) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K(+) dependent. The current-voltage relationship was slightly inwardly rectifying with a reversal potential of -52 +/- 2 mV, and the P(K)/P(Na) ratio was 14, indicating high selectivity for K(+). Concentration-response relationships for apamin and dequalinium had IC(50) values of 0.5 nm and 1.8 microm, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 microm BAPTA-AM completely blocked the effects of UTP on K(+) channel activation, indicating that the apamin-sensitive current was also Ca(2+) dependent. Moreover, channel activation was blocked by calmidazolium (IC(50) = 5 microm), suggesting a role for calmodulin in Ca(2+)-dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17beta produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K(+) channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca(2+) near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation.

Abnormal features in mutant cerebellar Purkinje cells lacking junctophilins

Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca 2+-mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase Cγ in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice.

Letter Regarding Article by Darbar et al, “Unmasking of Brugada Syndrome by Lithium”

To the Editor: It was with great interest that we read the study by Darbar and colleagues in a recent issue of Circulation .1 The authors reported on 2 patients who developed the Brugada ECG pattern after administration of lithium, a commonly used drug not previously reported to block cardiac Na+ channels. Surprisingly, Darbar and colleagues found that LiCl caused a concentration-dependent block of peak INa, with an IC50 of 6.8±0.4 μmol/L, a level 100 times below the therapeutic range (&1 mmol/L). They concluded that lithium is a potent blocker of cardiac Na+ channels and may unmask patients with the Brugada syndrome. It is well known from numerous studies (conducted during the last 3 decades) that Li+ ions permeate Na+ channels, as well as do Na+ ions, in a variety of excitable cell …

Regulation of Ca V 3.1 T‐type Ca 2+ channel expression by cAMP in pulmonary microvascular endothelial cells.

Activation of CaV3.1 T-type Ca2+ channel contributes to Gq-linked agonist-induced and store-operated Ca2+ entry in pulmonary microvascular endothelial cells (PMVECs). Since cAMP-dependent pathways have been implicated in upregulation of ion channels including voltage-gated Ca2+ channels in excitable cells, we sought to examine whether a sustained elevation of cAMP increases the synthesis of CaV3.1 channels in endothelial cells. Cultured rat PMVECs were exposed to exogenous cAMP (pCPT-cAMP 200 μM) for up to 96 hr and then subjected to whole-cell patch-clamp recordings to assess macroscopic T-type currents. Using a two-step voltage protocol to detect the maximally evoked tail current, we observed that exposure of exogenous cAMP did not have an immediate effect on the T-type current, whereas long-term exposure (> 24 hr) markedly increased the occurrence of high T-type current in PMVECs. The increased T-type current displayed virtually identical activation and inactivation properties as the original T-type current, and correlated with an elevated mRNA level after 24 hr exposure to exogenous cAMP. Further, we revealed that 8CPT-2Me-cAMP, a selective cAMP-regulated guanine nucleotide exchange factor (Epac) agonist, did not reproduce the effect of pCPT-cAMP on T-type current, while PKA inhibitor H89 and Rp-cAMPS, a competitive antagonist of cAMP binding to PKA, prevented the action of pCPT-cAMP. Together, these results support the idea that PKA-dependent signaling pathways are involved in up-regulation of CaV3.1 T-type Ca2+ channels in pulmonary microvascular endothelial cells. Supported by HL074116

Photochemical tools for remote control of ion channels in excitable cells

Various strategies have been developed recently for imparting light sensitivity onto normally insensitive cells. These include expression of natural photosensitive proteins, photolysis of caged agonists of native cell surface receptors and photoswitching of isomerizable tethered ligands that act on specially engineered ion channels and receptor targets. The development of chemical tools for optically stimulating or inhibiting signaling proteins has particular relevance for the nervous system, where precise, noninvasive control is an experimental and medical necessity.

Contribution of different Ca2+‐activated K+ channels to the first phase of the response to TRH in clonal rat anterior pituitary cells

Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca(2+) level ([Ca(2+)](i)), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca(2+)-activated K(+) channels (K(Ca)). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH(4) cells was assessed. The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca(2+) microfluoromety (fura-2). The involvement of different K(Ca) channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). Application of 100 nM iberiotoxin, 1 microM apamin, and 10 microM clotrimazole reduced the peak value of the outward K(+) current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. All the three main types of K(Ca) channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.

How to read clinical journals: XII. How you too can profit from pharmaceutical advertisements

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (∼0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Sodium channel toxins and neurotransmitter release.

Voltage-dependent sodium channels (VDSC) are an important class of ion channels in excitable cells, where they are responsible for the generation and conduction of action potential. In addition, the release of neurotransmitters from nerve terminals is influenced by sodium channel activity. The function of VDSC is subject to modulation by various neurotoxins, such as scorpion toxins, which have long been used as tools in the investigation of neurotransmitter release. This opens an interesting perspective concerning modulation of neurotransmission via pharmacological manipulation of sodium channel properties, which can lead to a better understanding of their physiological and pathological roles. Here we briefly review the studies of neurotoxins acting on sodium channels, focusing primarily on the view of the mechanisms of neurotransmitter release.

Tracking sodium channels in live cells: confocal imaging using fluorescently labeled toxins

One particularly important class of ion channels in excitable cells are the voltage-dependent sodium channels (VDSC). Knowledge of the distribution of VDSC in living cells is important for studies of neuronal excitability, development, and plasticity. Here, we demonstrate a new method for visualizing the spatial distribution of VDSC in living cells. To illustrate the capabilities of the technique, the distribution of VDSC in GH3 cells was revealed with fluorescent derivatives of the α-type and β-type scorpion toxins in conjunction with laser scanning confocal microscopy. Cells exhibited fluorescent hot spots on the surface of the membrane. This characteristic staining pattern was prevented by pre-incubation with unlabeled native toxins and blocked by membrane depolarization for α-type toxins. Labeling was not observed in cells lacking sodium channels (HEK 293) after incubation with fluorescent-labeled toxins. Image processing techniques were applied to identify the location of each cluster of labeled VDSC in these cells. The proposed method eliminates artefacts commonly introduced during sample preparation for immunostaining and should prove to be a valuable research tool for investigating VDSC distribution in living specimens.

Characterisation of the effects of potassium channel modulating agents on mouse intestinal smooth muscle.

The actions of agents which modulate ATP-sensitive potassium (K(ATP)) channels in excitable cells were investigated in an in-vitro preparation of mouse ileum from which the mucosa was removed. A range of potassium channel openers of diverse structure, cromakalim (0.1-100 microM), pinacidil (0.1-200 microM) and its analogue P1060 (0.1-200 microM), SDZ PCO400 ((-)-(3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent-1-enyloxy)-2H-1-benzopyran-6-carbonitrile) (0.3-60 microM), caused concentration-related reduction in twitch height of electrical field stimulated ileum. P1060 and SDZ PCO400 were the most potent agents; diazoxide (0.1-100 microM) was without effect. The order of inhibitory potency, based on EC50 values (concentration of a relaxant producing 50% of the maximum inhibition of twitch) was: P1060 = SDZ PCO400 > cromakalim > pinacidil. The relaxant effect of the potassium channel openers was antagonised by the sulfonylureas glibenclamide (0.1-1.0 microM) and glipizide (3-30 microM) but the nature of the antagonism differed. Antagonism of P1060 and SDZ PCO400 by glibenclamide appeared to be competitive whereas the antagonism of relaxation induced by cromakalim and pinacidil was apparently not competitive. Both phentolamine (1-10 microM) and tolbutamide (100-300 microM) showed competitive antagonism of the actions of pinacidil while yohimbine (1-20 microM) did not antagonise relaxation and appeared to have actions at sites other than the K(ATP) channel in this preparation. The relative effectiveness of the antagonists on pinacidil-induced relaxation was found to be: glibenclamide > phentolamine > tolbutamide > yohimbine, which is in agreement with studies in other tissues. The results show that many structurally diverse potassium channel openers are potent relaxants of mouse ileum. These observations are consistent with the existence of ATP-dependent K+ channels in murine intestinal muscle which, however, differ somewhat in properties from those reported for vascular muscle and pancreatic beta-cells.

The purine degradation pathway: Possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1

The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO 2 and NH 4 + or recycled in the primary metabolism through the purine degradation pathway.

Long-term enhancement of synaptic transmission induced by veratridine in rat CA3 hippocampal neurons.

Veratridine is a neurotoxin that induces persistent activation of sodium channels in excitable cells. We investigated the effects of this toxin on excitatory synaptic transmission in CA3 neurons of juvenile rat hippocampus using whole-cell patch-clamp and field-potential recordings. The population spikes evoked by electrical stimulation of the mossy fiber were gradually enhanced after washout of veratridine (0.3 microM), but they were not enhanced by the co-application of veratridine and an N-methyl-D-aspartate (NMDA) receptor antagonist (D-APV, 30 microM). When a pipette solution contained QX-314 that antagonized the effect of veratridine in the recorded neuron, oscillatory membrane depolarization appeared in the early stage during bath-application of veratridine and gradually decreased in the late stage. After washout of veratridine, however, the oscillatory depolarization was gradually restored and maintained for at least 3 h. This oscillatory depolarization was also abolished by D-APV. We suggest that the activation of NMDA receptors is involved in the veratridine-induced long-lasting enhancement in the excitatory synaptic transmission in rat CA3 hippocampal neurons.

Subunit Composition Determines Kv1 Potassium Channel Surface Expression

Shaker-related or Kv1 voltage-gated K(+) channels play critical roles in regulating the excitability of mammalian neurons. Native Kv1 channel complexes are octamers of four integral membrane alpha subunits and four cytoplasmic beta subunits, such that a tremendous diversity of channel complexes can be assembled from the array of alpha and beta subunits expressed in the brain. However, biochemical and immunohistochemical studies have demonstrated that only certain complexes predominate in the mammalian brain, suggesting that regulatory mechanisms exist that ensure plasma membrane targeting of only physiologically appropriate channel complexes. Here we show that Kv1 channels assembled as homo- or heterotetrameric complexes had distinct surface expression characteristics in both transfected mammalian cells and hippocampal neurons. Homotetrameric Kv1.1 channels were localized to endoplasmic reticulum, Kv1.4 channels to the cell surface, and Kv1.2 channels to both endoplasmic reticulum and the cell surface. Heteromeric assembly with Kv1.4 resulted in dose-dependent increases in cell surface expression of coassembled Kv1.1 and Kv1.2, while coassembly with Kv1.1 had a dominant-negative effect on Kv1.2 and Kv1.4 surface expression. Coassembly with Kv beta subunits promoted cell surface expression of each Kv1 heteromeric complex. These data suggest that subunit composition and stoichiometry determine surface expression characteristics of Kv1 channels in excitable cells.

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.

Batrachotoxin-Resistant Na + Channels Derived from Point Mutations in Transmembrane Segment D4-S6

Local anesthetics (LAs) block voltage-gated Na + channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na + channel α-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na + channels ( μ1/rSkm1). We show that μ1-F1579K and μ1-N1584K channels become completely resistant to 5 μM BTX. In contrast, μ1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.

A novel capacitative calcium entry channel expressed in excitable cells.

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

Functional Anatomy of Scorpion Toxins Affecting Sodium Channels

AbstractLong chain scorpion toxins (made of 60 to 70 amino acids) acting on voltage-gated sodium channels in excitable cells are responsible for human envenomation, and comprise a-toxins that inhibit sodium current inactivation and p-toxins, that modify the activation process. These toxins may be further divided according to their pharmacological activities. Thus, a-toxins highly active on mammals or insects, as well as α like toxins may be distinguished within the α-toxin class. The β-toxin class includes toxins active on mammals and, as a separate group, the excitatory and depressant toxins active exclusively on insects. All these toxins possess 4 disulfide bridges and share 15 similar non cystine residues. Accordingly, their 3D structure is highly conserved, comprising an α-helix and a triple stranded β-sheet. The most solvent exposed turns of this structure are prone to insertions or deletions, and accordingly correspond to the most structurally variable regions of the toxins. They have been tested by...

20] Calcium influx during an action potential

Action potentials open voltage-sensitive calcium channels in excitable cells, leading to an influx of calcium ions. Calcium ions may control, among others, cell excitability, neurotransmitter release, or gene transcription. This chapter discusses two different methods that can be used to quantify the Ca 2+ flows into a cell or cell compartment during an action potential. Quantification of Ca 2+ influx may be a first step toward the construction of a model for the calcium dynamics of cells. It may also serve as a reference to study pathological processes such as cell death during ischemia or amyotrophic lateral sclerosis, where increases in calcium influx have generally been implicated. The two methods that are compared in this chapter are a voltage-clamp method, in which cells are voltage clamped with an action potential waveform command, and a fluorescence method, in which cells are loaded with a high concentration of a calcium-sensitive fluorescent dye. Both methods are applied to a giant axosomatic terminal, called the “calyx of Held,” in a slice preparation of the rat brain stem. Each principal cell in the medial nucleus of the trapezoid body (MNTB) is contacted by a single calyx-type terminal. This synapse is part of a fast auditory pathway that is involved in the localization of sound. This chapter is interested in the relationship between calcium influx and neurotransmitter release at this glutamatergic synapse. Both methods gave similar values for the number of calcium ions that enter the presynaptic terminal during an action potential. It first discusses the action potential waveform voltage-clamp method.