Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

ω-Aga IVA selectively inhibits the calcium-dependent fraction of the evoked release of [3H]GABA from synaptosomes

The effect of -Aga IVA, a P-type Ca²⁺ channel blocker, on the release of the inhibitory neurotransmitter GABA and on the elevation of Ca_i induced by depolarization was investigated in [³H]GABA and fura-2 preloaded mouse brain synaptosomes, respectively. Two strategies (i.e. 20 mM external K⁺ and veratridine) that depolarize by different mechanisms the preparation were used. High K⁺ elevates Ca_i and induces [³H]GABA release in the absence of external Na⁺ and in the presence of TTX, conditions that abolish veratridine induced responses. The effect of -Aga IVA on the Ca²⁺ and Na⁺ dependent fractions of the depolarization evoked release of [³H]GABA were separately investigated in synaptosomes depolarized with high K⁺ in the absence of extermal Na⁺ and with veratridine in the absence of external Ca²⁺, respectively. The Ca²⁺ dependent fraction of the evoked release of [³H]GABA and the elevation of Ca²⁺ induced by high K⁺ are markedly inhibited (about 50%) in synaptosomes exposed to -Aga IVA (300 nM) for 3 min before depolarization, whereas the Na⁺ dependent, Ca²⁺ independent carrier mediated release of [³H]GABA induced by veratridine, which is sensitive to verapamil and amiloride, is not modified by -Aga IVA. Our results indicate that an -Aga IVA sensitive type of Ca²⁺ channel is highly involved in GABA exocytosis.     

Aminopyridines Potentiate Synaptic and Neuromuscular Transmission by Targeting the Voltage-activated Calcium Channel �� Subunit

Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K⁺ (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca²⁺ channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, α-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca²⁺ currents primarily through the intracellular β₃ subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.     

Bisphenol A Modulates Calcium Currents and Intracellular Calcium Concentration in Rat Dorsal Root Ganglion Neurons

The endocrine-disrupting chemical bisphenol A (BPA) is used to manufacture plastics including food containers, and it may leach into these containers. Consumption of BPA that has leached out of plastics may be harmful as recent research highlighted that BPA can induce alterations in the nervous system. In the present work, we studied the effects of BPA on Ca²⁺ channels in dorsal root ganglion (DRG) neurons. Using whole-cell patch-clamp recordings, we found that I _Ca could be reduced by BPA in a concentration-dependent manner. Additionally, BPA shifted the activation curve of calcium currents toward a depolarizing direction and increased the slope factor of the curve. The inactivation curve for the currents was also assessed, and the curve shifted toward the depolarizing direction, although it was not significant. Moreover, inhibitory effects of BPA on the increments of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by 50 mM KCl were observed in DRG neurons using a laser scanning confocal microscopy assay. Further work revealed that the PKA and PKC pathways may be involved in the inhibitory effects of BPA since the PKA antagonist GÖ-6983 and the PKC antagonist H-89 significantly alleviated the inhibitory effects of BPA on I _Ca. As such, the results of the present study provide direct evidence that BPA decreases I _Ca and impairs calcium homeostasis, which may be involved in any toxic effects of BPA on DRG neurons.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Monovalent amidiniums block calcium channels in chick sensory neurons

The effect of amidiniums on high-threshold Ca²⁺ channel currents (I _Ca) was studied in chick dorsal root ganglion neurons. Guanidinium reduced I _Ca in a dose-dependent fashion. The block was relieved by increasing the concentration of the permeant ions, Ba²⁺ or Ca²⁺, suggesting a competition for a common binding site within the channel. Formamidinium and methyl-guanidinium suppressed I _Ca with similar potencies, whereas l-arginine had no effect. A neutral amidine, urea, increased I _Ca. In Ca²⁺-free solutions guanidinium and Na⁺ permeated through the Ca²⁺ channel equally well. Structure-activity relationship obtained for blocking efficacies of different amidiniums are used to discuss possible configurations of the selectivity filter in the Ca²⁺ channel.The author wishes to thank Ms. S. Engers for the cell isolation and making of the electrodes.     

Passive Glial Cells, Fact or Artifact?

Astrocytes that are recorded in acute tissue slices of rat hippocampus using whole-cell patch-clamp, commonly exhibit voltage-activated Na⁺ and K⁺ currents. Some reports have described astrocytes that appear to lack voltage-activated currents and proposed that these cells constitute a subpopulation of electrophysiologically passive astrocytes. We show here that these cells can spontaneously change during a recording unmasking expression of previously suppressed voltage-activated currents, suggesting that such cells do not represent a subpopulation of passive astrocytes. Superfusion of a low Ca²⁺/EGTA solution was able to reversibly suppress voltage-activated K⁺ currents in cultured astrocytes. Currents were restored upon addition of normal bath Ca²⁺. These effects of Ca²⁺ on both outward and inward K⁺ currents were dose- and time-dependent, with increasing concentrations of Ca²⁺ (from 0 to 800 μm) leading to a gradual unmasking of voltage-dependent outward and inward K⁺ currents. The transition from an apparently passive cell to one exhibiting prominent voltage-activated currents was not associated with any changes in membrane capacitance or access resistance. By contrast, in cells in which low access resistance or poor seal accounted for the absence of voltage-activated currents, improvement of cell access was always accompanied by changes in series resistance and membrane capacitance. We propose that spillage of pipette solution containing low Ca²⁺/EGTA during cell approach in slice recordings and/or poor cell access, lead to a transient masking of voltage-activated currents even in astrocytes that express prominent voltage-activated currents. These cells, however, do not constitute a subpopulation of electrophysiologically passive astrocytes. Received: 22 April 1998/Revised: 8 September 1998     

Venom from Anemesia species of spider modulates high voltage-activated Ca(2+) currents from rat cultured sensory neurones and excitatory post synaptic currents from rat hippocampal slices

The actions of crude venom from Anemesia species of spider were investigated in cultured dorsal root ganglion neurones from neonatal rats and hippocampal slices. Using mass spectrometry (MALDI-TOF MS), 10-12 distinct peptides with masses between about 3 and 10kDa were identified in the crude spider venom. At a concentration of 5 microg/ml crude Anemesia venom transiently enhanced the mean peak whole cell voltage-activated Ca(2+) current in a voltage-dependent manner and potentiated transient increases in intracellular Ca(2+) triggered by 30mM KCI as measured using Fura-2 fluorescence imaging. Additionally, 5-8 microg/ml Anemesia venom increased the amplitude of glutamatergic excitatory postsynaptic currents evoked in hippocampal slices. Omega-Conotoxin GVIA (1 microM) prevented the increase in voltage-activated Ca(2+) currents produced by Anemesia venom. This attenuation occurred when the cone shell toxin was applied before or after the spider venom. Anemesia venom (5 microg/ml) created no significant change in evoked action potentials but produced modest but significant inhibition of voltage-activated K(+) currents. At a concentration of 50 microg/ml Anemesia venom only produced reversible inhibitory effects, decreasing voltage-activated Ca(2+) currents. However, no significant effects on Ca(2+) currents were observed with a concentration of 0.5 microg/ml. The toxin(s) in the venom that enhanced Ca(2+) influx into sensory neurones was heat-sensitive and was made inactive by boiling or repetitive freeze-thawing. Boiled venom (5 microg/ml) produced significant inhibition of voltage-activated Ca(2+) currents and freeze-thawed venom inhibited Ca(2+) transients measured using Fura-2 fluorescence. Our data suggest that crude Anemesia venom contains components, which increased neuronal excitability and neurotransmission, at least in part this was mediated by enhancing Ca(2+) influx through N-type voltage-activated Ca(2+) channels.     

Combined actions of Pb2+, Zn2+, and Al3+ on voltage-activated calcium channel currents

Summary 1. Whole-cell patch clamp experiments were performed on rat dorsal root ganglion (DRG) neurons to investigate the actions of various combinations of Pb²⁺, Zn²⁺, and Al³⁺ on voltage-activated calcium channel currents (VACCCs).2. Each of these metals has been shown to reduce VACCCs.3. We investigated the effects of simultaneous application of two cations in the range of their IC₅₀ values. For all possible combinations (Pb²⁺/Zu²⁺, Zn²⁺/Al³⁺, Al³⁺/Pb²⁺), independent of the order of application, we found additive actions on VACCCs.4. We observed a 75% (±9%) block of the control current when two cations were applied simultaneously. This observation is consistent with both, an action of two metals at the same site as well as independent actions at different locations of the ion channel.5. The additivity of the effects should be taken into account for questions of public health and the assessment of threshold limits in cases of environmental contamination.     

Effects of neuropathy on high-voltage-activated Ca current in sensory neurones

Voltage-dependent Ca²⁺ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the Ca_V2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca²⁺ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from Ca_V2.3(−/−) knock-out and wild-type (WT) mice. DRG neurones from Ca_V2.3(−/−) mice had significantly reduced sensitivity to SNX-482 versus WT mice. DRGs from Ca_V2.3(−/−) mice also had increased sensitivity to the Ca_V2.2 VDCC blocker ω-conotoxin. In WT mice, PSNL caused a significant increase in ω-conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In Ca_V2.3(−/−) mice, PSNL caused a significant reduction in ω-conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca²⁺ current were not accompanied by effects on voltage-dependence of activation in either Ca_V2.3(−/−) or WT mice. These data suggest that Ca_V2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca²⁺ current in these cells. In WT mice, PSNL caused adaptive changes in Ca_V2.2- and Ca_V2.3-mediated Ca²⁺ currents, supporting roles for these VDCCs in nociception during neuropathy. In Ca_V2.3(−/−) mice, PSNL-induced changes in Ca_V1 and Ca_V2.2 Ca²⁺ current, consistent with alternative adaptive mechanisms occurring in the absence of Ca_V2.3 subunits.     

Neurohormone D induces ionic current changes in cockroach central neurones

The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), increases the frequency of spontaneous activity in dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana. The increase in spike frequency is accompanied by changes in the shape and the amplitude of the single action potentials, e.g. a more pronounced afterhyperpolarization. Effects of NHD on membrane currents were investigated in these DUM cells with whole-cell voltage-clamp measurements. A voltage-independent Ca²⁺ current flowing at the resting potential (I_Ca,R) was found. NHD, at nanomolar concentrations, enhanced this I_Ca,R in a concentration-dependent manner. 0.1 mM Cd²⁺markedly reduced I_Ca,R and in this case I_Ca,R was hardly potentiated by NHD.In the presence of NHD a fast activating Ca²⁺-dependent K⁺current sensitive to charybdotoxin and to low concentrations of tetraethylammonium was augmented. The enhanced afterhyperpolarization of action potentials can be accounted for by the increase in the Ca²⁺-dependent K⁺ current.The changes of the membrane currents induced by NHD are discussed with respect to further effects on the spike pattern and in relation to the previously described mode of action of AKH-peptides in other preparations.Abbreviations NHD neurohormone D - AKH adipokinetic hormone     

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

Low voltage-activated (LVA) T-type Ca²⁺ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca²⁺ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Ca_v3.1 to Ca_v3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca²⁺ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Ca_v3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Ca_v3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Ca_v3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca²⁺ channels.     

Characterization of the type of calcium channel primarily regulating GABA exocytosis from brain nerve endings

In an attempt to further characterize the type of Ca²⁺ channels primarily regulating GABA exocytosis, the effects of increasing concentrations of CTx MVIIC,--Aga IVA and other Ca²⁺ channel blockers (nitrendipine, Cd²⁺ and Ni²⁺), commonly used for pharmacologically discerning among the various types of Ca²⁺ channels, were tested on the dissected Ca²⁺ dependent fraction of the depolarization evoked release of GABA from mouse brain synaptosomes. Our results show that -CTx MVIIC inhibits GABA exocytosis with a calculated IC₅₀ of 3 M and -Aga IVA with a calculated IC₅₀ of 50 nM. The divalent cation Cd²⁺ only diminishes GABA exocytosis at 70 M, but does not modify this response at lower concentrations (i.e. 1 and 10 M). Neither nitrendipine (10 M) nor Ni²⁺ (100 M and 500 M) modified GABA exocytosis. The failure of nitrendipine at a high concentration to inhibit GABA exocytosis discards L-type Ca²⁺ channels as the main regulators of this response; likewise that of Ni²⁺ discards Ca²⁺ channels of the N-type, and the failure of nM concentrations of -CTx MVIIC or 500 M Ni²⁺, also discards alpha_1A/Q-type Ca²⁺ channels as the main regulators of the GABA response. On the basis of these results and in particular of the higher potency of -Aga IVA than -CTx MVIIC, it is concluded that the type of Ca²⁺ channels that primarily determine the exocytosis of GABA belong to a P-like type of Ca²⁺ channels.     

Physiological and Biochemical Analysis to Reveal the Molecular Basis for Black Widow Spiderling Toxicity

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD₅₀ being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve‐hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage‐activated Na⁺, K⁺, and Ca²⁺ channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity.     

Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca²⁺ channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca²⁺ channels. The NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca²⁺ channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.     

Inhibition of Ca2+-dependent Cl− channels from secretory epithelial cells by low internal pH

The actions of intracellular pH (pH _i ) on Ca²⁺dependent Cl^? channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca²⁺dependent Cl^? channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca²⁺] _i ) or by using pipette solutions with buffered Ca²⁺ levels. Large currents were activated in T84 and parotid cells by both methods with pH _i levels of 7.3 or 8.3. Little or no Cl^? channel current was activated with pH _i at 6.4. We used on-cell patch clamp methods to investigate the actions of low pH _i on single Cl^? channel current amplitude in T84 cells. Lowering the pH _i had little or no effect on the current amplitude of a 8 pS Cl^? channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca²⁺-activated Cl^? channels.     

Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca²⁺, although the absolute magnitude of the Ca²⁺ current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca²⁺ to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca²⁺ was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca²⁺-sensing dye Fluo-4, and found that acidification increased [Ca²⁺]_i in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca²⁺, our single cell data strongly challenges the view that Ca²⁺ entry through the ASIC1a receptor itself contributes to this response.