Diabetic neuropathy enhances voltage-activated Ca2+ channel activity and its control by M4 muscarinic receptors in primary sensory neurons

Painful neuropathy is one of the most serious complications of diabetes and remains difficult to treat. The muscarinic acetylcholine receptor (mAChR) agonists have a profound analgesic effect on painful diabetic neuropathy. Here we determined changes in T-type and high voltage-activated Ca(2+) channels (HVACCs) and their regulation by mAChRs in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathy. The HVACC currents in large neurons, T-type currents in medium and large neurons, the percentage of small DRG neurons with T-type currents, and the Cav3.2 mRNA level were significantly increased in diabetic rats compared with those in control rats. The mAChR agonist oxotremorine-M significantly inhibited HVACCs in a greater proportion of DRG neurons with and without T-type currents in diabetic than in control rats. In contrast, oxotremorine-M had no effect on HVACCs in small and large neurons with T-type currents and in most medium neurons with T-type currents from control rats. The M(2) and M(4) antagonist himbacine abolished the effect of oxotremorine-M on HVACCs in both groups. The selective M(4) antagonist muscarinic toxin-3 caused a greater attenuation of the effect of oxotremorine-M on HVACCs in small and medium DRG neurons in diabetic than in control rats. Additionally, the mRNA and protein levels of M(4), but not M(2), in the DRG were significantly greater in diabetic than in control rats. Our findings suggest that diabetic neuropathy potentiates the activity of T-type and HVACCs in primary sensory neurons. M(4) mAChRs are up-regulated in DRG neurons and probably account for increased muscarinic analgesic effects in diabetic neuropathic pain.     

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.     

Protein kinase CK2 contributes to diminished small conductance Ca2+‐activated K+ channel activity of hypothalamic pre‐sympathetic neurons in hypertension

Small conductance calcium‐activated K⁺ (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up‐regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre‐sympathetic neurons in SHRs. Perforated whole‐cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar‐Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin‐sensitive SK currents and depolarization‐induced medium after‐hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after‐depolarization potential of PVN neurons in SHRs. In addition, CK2‐mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2‐mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre‐sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre‐sympathetic neurons and increased sympathetic vasomotor tone in hypertension.

     

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.     

Membrane stretch augments the cardiac muscarinic K+ channel activity

Arachidonic acid has been shown to activate K⁺-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (G _K )-gated, muscarinic K⁺ (K_ACh) channel can also be activated by arachidonic acid, we investigated whether the K_ACh channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), K_ACh channels were not active, and negative pressure failed to activate these channels. With ACh (10 m) in the pipette, applying negative pressure (0 to –80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and inside-out patches (100 m GTP in bath). Membrane stretch did not alter the sensitivity of the K_ACh channel to GTP. When G _K was maximally activated with 100 m GTPS in inside-out patches, the K_ACh channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the K_ACh channel in the absence of ACh and GTP; K_ACh channel activity was further increased by stretch. These results indicate that the atrial muscarinic K⁺ channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.     

Inhibition of Ca2+ and K+ channels in sympathetic neurons by neuropeptides and other ganglionic transmitters.

Neuropeptides are known to modulate the excitability of frog sympathetic neurons by inhibiting the M-current and increasing the leak current, but their effects on Ca2+ channels are poorly understood. We compared effects of LHRH, substance P, epinephrine, and muscarine on Ca2+, K+, and leak currents in dissociated frog sympathetic neurons. At concentrations that inhibit M-current, LHRH and substance P strongly reduced N-type Ca2+ current and induced a leak conductance that may contribute to slow EPSPs. In contrast, muscarine produced little reduction of Ca2+ current, even in cells in which it strongly suppressed the M-current. We find that peptidergic inhibition of Ca2+ channels involves G proteins, but does not require protein kinases. In addition, it leads to reductions in Ca2(+)-activated K+ current and catecholamine release.     

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult (P < 0.05) and that these were predominantly due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation (V(1/2)) yielded linear and parallel relationships, and the change in V(1/2) for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 +/- 2-mV depolarization for adult myocytes and for an 18 +/- 1-mV depolarization for fetal myocytes (P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 microM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.     

Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Hexamethonium increases the excitability of sympathetic neurons by the blockade of the Ca2+-activated K+ channels

Effects of hexamethonium (C6) on the excitability of sympathetic ganglion cells were examined by means of intracellular recording. When DC currents were injected, high concentrations of C6 significantly augmented the repetitive firing of the cells without any change in threshold voltage for initiation of the spike. The Ca2+-sensitive component of the after-hyperpolarization following a spike was reduced by C6 in a dose-dependent fashion (0.3 to 10 mM). C6 slightly affected parameters for the spike but neither the resting membrane potential nor the input membrane resistance. The amplitude of the Ca2+ spike (in the presence of 1 microM tetrodotoxin and 10 mM tetraethylammonium) was not affected even by 30 mM C6. Bee venom (0.3 micrograms/ml) which contains apamin showed similar effects. These results suggest that C6 blocks the Ca2+-activated K+ channels, resulting in an increase in excitability of the cells.     

Control of the cardiac muscarinic K+ channel by beta-arrestin 2

Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Ca2+ entry via AMPA-type glutamate receptors triggers Ca2+-induced Ca2+ release from ryanodine receptors in rat spiral ganglion neurons

Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.     

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.     

Snake toxins that bind specifically to individual subtypes of muscarinic receptors

This paper discusses the properties of the three most specific ligands found for the extracellular faces of M1, M2 and M4 muscarinic receptors (m1-toxin1, m2-toxin and m4-toxin, respectively). The primary goal of this paper is to show the known and potential usefulness of these toxins and their biotinylated, radioactive, fluorescent and mutated derivatives.     

Ca2+-activated chloride channel activity during Ca2+ alternans in ventricular myocytes

Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca²⁺-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl^? channel blocker DIDS or lowering external Cl^? concentration identifying it as a Ca²⁺-activated Cl^? current (I_CaCC). The data suggest that I_CaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.     

Analysis of a Ca2+-activated K+ channel that mediates hyperpolarization via the thrombin receptor pathway

Dami human leukemia cells express G protein-coupled thrombinreceptors that operate through the phospholipase C pathway. When thesereceptors are activated by -thrombin or by thrombinreceptor-activating peptide, an elevation in cytosolicCa²⁺ concentration develops thatis accompanied by hyperpolarization of the plasma membrane. Thistransitory phase of hyperpolarization is primarily mediated by inwardlyrectifying, Ca²⁺-activatedK⁺ channels that have an inwardconductance of ~24 pS. In cell-attached patches the channels openwithin seconds after superfusion of the cell with thrombinreceptor-activating peptide. In inside-out patches, perfusion ofsubmicromolar Ca²⁺ onto thecytosolic surface of the membrane is sufficient to activate thechannels. In outside-out patches, channel opening can be blocked bynanomolar concentrations of charybdotoxin. The function of theseintermediate-sized inwardly rectifying,Ca²⁺-activatedK⁺ channels has not beenestablished; however, by analogy with other cell systems, they mayserve to regulate cell volume during cellular activation or to increasethe electromotive drive that sustains Na⁺ and/orCa²⁺ influx through ligand-gatedcation channels.

     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.