P-type Calcium Channels Research Articles

The Role of Kv1.2-Containing Potassium Channels in Serotonin-Induced Glutamate Release from Thalamocortical Terminals in Rat Frontal Cortex

Serotonin 5-HT(2A) receptors have been implicated in psychiatric illness and the psychotomimetic effects of hallucinogens. In brain slices, focal stimulation of 5-HT(2A) receptors in rat prefrontal cortex results in dramatically increased glutamate release onto layer V pyramidal neurons, as measured by an increase in "spontaneous" (nonelectrically evoked) EPSCs. This glutamate release is blocked by tetrodotoxin (TTX) and is thought to involve local spiking in thalamocortical axon terminals; however, the detailed mechanism has remained unclear. Here, we investigate parallels in EPSCs induced by either serotonin or the potassium channel blockers 4-aminopyridine (4-AP) or alpha-dendrotoxin (DTX). DTX, a selective blocker of Kv1.1-, Kv1.2-, and Kv1.6-containing potassium channels, has been shown to release glutamate in cortical synaptosomes, presumably by inhibiting a subthreshold-activated, slowly inactivating potassium conductance. By comparing DTX with other potassium channel blockers, we found that the ability to induce EPSCs in cortical pyramidal neurons depends on affinity for Kv1.2 subunits. DTX-induced EPSCs are similar to 5-HT-induced EPSCs in terms of sensitivity to TTX and omega-agatoxin-IVA (a blocker of P-type calcium channels) and laminar selectivity. The involvement of thalamocortical terminals in DTX-induced EPSCs was confirmed by suppression of these EPSCs by micro-opiates and thalamic lesions. More directly, DTX-induced EPSCs substantially occlude those induced by 5-HT, suggesting a common mechanism of action. No occlusion by DTX was seen when EPSCs were induced by a nicotinic mechanism. These results indicate that blockade of Kv1.2-containing potassium channels is part of the mechanism underlying 5-HT-induced glutamate release from thalamocortical terminals.

Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model for neuropathy. (Department of Pharmacology, University College London, London, United Kingdom) Pain. 2001;92:233-246.

Neuropathic pain, due to peripheral nerve damage, can include allodynia (perception of innocuous stimuli as being painful), hyperalgesia (increased sensitivity to noxious stimuli) and spontaneous pain, often accompanied by sensory deficits. Plasticity in transmission and modulatory systems are implicated in the underlying mechanisms. The Kin and Chung rodent model of neuropathy employed involved unilateral tight ligation of 2 (L5 and L6) of the 3 (L4, L5, and L6) spinal nerves of the sciatic nerve and reproducibly induced mechanical and cold allodynia in the ipsilateral hindpaw over the 14 day postoperative period. In vivo electrophysiological techniques have then been used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation (SNL). Activation of voltage-dependent calcium channels (VDCCs) is critical for neurotransmitter release and neuronal excitability, and antagonists can be antinociceptive. Here, for the first time, the effect of N-type and P-type VDCC antagonists (ω-conotoxin-GVIA and ω-agatoxin-IVA, respectively) on the evoked dorsal horn neuronal responses after neuropathy have been investigated. Conclude both presynaptic and postsynaptic VDCCs appear to be important.

Decreased calcium influx into the neonatal rat motor nerve terminals can recruit additional neuromuscular junctions during the synapse elimination period

Individual skeletal muscle fibers in newborn vertebrates are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in an activity-dependent process of synaptic elimination by axonal competition. Because synaptic activity depends strongly on the influx of calcium from the external media via presynaptic voltage-dependent calcium channels, we investigate the relationship between calcium channels, synaptic activity and developmental axonal elimination. We studied how several calcium channel blockers affect (after 1 h of incubation) the total number of functional axons per muscle fiber (poly-innervation index) of the Levator auris longus muscle of 6-day-old rats. We determined the poly-innervation index by gradually raising the stimulus amplitude and recorded the recruitment of one or more axons that produced a stepwise increment of the endplate potential. The L-type channel blocker nitrendipine (1 μM) increased the mean poly-innervation index (35.79%±3.91; P<0.05). This effect was not washed out with normal Ringer, although the poly-innervation index returned to the control value when high-calcium Ringer (5 mM) was used. The P-type channel blocker ω-agatoxin-IVA (100 nM) also increased the number of recruitable endplate potentials (27.49%±1.78; P<0.05), whereas N-type channel blocker ω-conotoxin-GVIA (1 μM) was ineffective ( P>0.05). However, neither nitrendipine nor ω-agatoxin-IVA modified the poly-innervation index on high-calcium Ringer ( P>0.05 in both cases). A more intense inhibition of calcium influx (by the sequential use of two calcium channel blockers) did not recruit any additional silent synapses. Moderately increasing the magnesium ions (by 500 μM) in the physiological solution produces a synaptic recruitment (36.78%±2.1; P<0.05) similar to that with L- and P-type calcium channel blockers incubation. This magnesium effect was not washed with normal Ringer but a Ringer that is high in calcium can reverse it. The recruited endings were identified by selective activity-dependent loading with styryl dyes. Rhodaminated α-bungarotoxin-labeled acetylcholine receptors were present in the postsynaptic counterpart. Based on these findings we suggest that, before their complete retraction, functionally silent nerve terminals can be manifested or recovered if calcium influx is reduced by a calcium channel blocker or if external magnesium is increased. The normal activation of this calcium-dependent silencing mechanism during development may be related to the final loss of the supernumerary axons.

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega-agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

Two distinct oscillatory states determined by the NMDA receptor in rat inferior olive

1. The effects of N-methyl-D-aspartate (NMDA) receptor activation and blockade on subthreshold membrane potential oscillations of inferior olivary neurones were studied in brainstem slices from 12- to 21-day-old rats. 2. Dizocilpine (MK-801), a non-competitive NMDA antagonist, at 1-45 microM abolished spontaneous subthreshold oscillations, without affecting membrane potential, input resistance, or the low-threshold calcium current, I(T). Ketamine (100 microM), a non-competitive NMDA antagonist, and L-689,560 (20 microM), an antagonist at the glycine site of the NMDA receptor, also abolished the oscillations, while the competitive non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20-50 microM) had no effect. 3. NMDA (100 microM) induced 4.1 Hz subthreshold oscillations and reversibly depolarized olivary neurones by 13.7 mV. In contrast, 10 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and 20 microM kainic acid depolarized the membrane equivalently but did not induce oscillations. 4. Both NMDA-induced and spontaneous subthreshold oscillations were unaffected by 1 microM tetrodotoxin and were prevented by substituting extracellular calcium with cobalt. 5. Removing magnesium from the perfusate did not affect spontaneous subthreshold oscillations but did prevent NMDA-induced oscillations. 6. NMDA-induced oscillations were resistant to 50 microM mibefradil, an I(T) blocker, in contrast to spontaneous oscillations. Both oscillations were inhibited by 20 microM nifedipine, an L-type calcium channel antagonist, and 200 nM omega-agatoxin IVA, a P-type calcium channel blocker. Bay K 8644 (10 microM), an L-type Ca(2+) agonist, significantly enhanced the amplitude of both spontaneous and NMDA-induced oscillations. 7. The data indicate that NMDA receptor activation induces olivary neurones to manifest high amplitude membrane potential oscillations in part mediated by L- and P- but not T-type calcium currents. Moreover, the data demonstrate that NMDA receptor currents are necessary for generation of spontaneous subthreshold oscillations in the inferior olive.

Aplysia Ror Forms Clusters on the Surface of Identified Neuroendocrine Cells

The ror receptors are a highly conserved family of receptor tyrosine kinases genetically implicated in the establishment of cellular polarity. We have cloned a ror receptor from the marine mollusk Aplysia californica. Aplysia ror (Apror) is expressed in most developing neurons and some adult neuronal populations, including the neuroendocrine bag-cell neurons. The Apror protein is present in peripheral neuronal processes and ganglionic neuropil, implicating the kinase in the regulation of growth and/or synaptic events. In cultured bag-cell neurons, the majority of the protein is stored in intracellular organelles, whereas only a small fraction is expressed on the surface. When expressed on the cell surface, the protein is clustered on neurites, suggesting that Apror is involved in the organization of functional domains within neurons. Apror immunoreactivity partially colocalizes with the P-type calcium channel BC-α1A at bag-cell neuron varicosities, suggesting a role for Apror in organizing neuropeptide release sites.

Modulating Action of Hyperforin on the P-Type Calcium Channels in the Membranes of Rat Cerebellar Purkinje Neurons

A modulating action of hyperforin (an active compound of the extract from Hypericum perforatum) on a high-threshold component of the calcium current, sensitive to application of 100 nM ω-Aga-IVA toxin and identified as P current, was studied on freshly isolated Purkinje neurons with the use of a patch-clamp technique in the whole-cell configuration. It was shown that extracellular application of 0.8 μM hyperforin caused a shift of the current-voltage (I-V) relationship of P current by -(8 ± 2) mV, slowdown of the activation kinetics, and a decrease in the amplitude of this current. The shift of the I-V relationship and slowdown of activation kinetics developed for less than 10 sec, while the P-current amplitude decreased for a much longer time (several minutes) and depended on the intracellular concentration of Ca2+ ions. ω-Aga-IVA toxin at the concentration of 100 nM completely blocked the recorded inward current in the presence of 0.8 μM hyperforin. In experiments with intracellular perfusion of Purkinje neurons, we found that interaction of hyperforin with its binding site occurs at the external side of the cell membrane. The study of the mechanisms involved in the hyperforin-induced P-current modulation revealed that 1 mM GTPγS (activating GTs proteins, as well as activating or blocking GMs proteins) or 1-2 mM GDPβS (blocking GTs and GMs proteins) in the intracellular solution did not affect the hyperforin-induced modulation of P current. Hyperforin-induced Ca2+-independent shift of the I-V relationship and slowdown of the activation kinetics of P current were abolished in the presence of 0.5 μM calmidazolium in the extracellular medium.

L to d Amino Acid Isomerization in a Peptide Hormone Is a Late Post-translational Event Occurring in Specialized Neurosecretory Cells

Modification of the chirality of a single amino acid residue within a peptide chain appears to be novel additional mechanism leading to structural and functional diversification of eukaryotic bioactive peptides. This phenomenon has been studied at the cellular level in a neuroendocrine organ which elaborates a mixture of diastereoisomers of a 72-residue neuropeptide, crustacean hyperglycemic hormone. For the first time, amino acid isomerization has been shown to occur in the perikarya of fully specialized neurosecretory cells, as a late step of the maturation of the hyperglycemic hormone precursor and after propeptide cleavage. The specificity and efficiency of this phenomenon indicates the existence of a new enzyme family involved in the biogenesis of peptide hormones.

A(2A) adenosine receptor facilitation of neuromuscular transmission: influence of stimulus paradigm on calcium mobilization.

The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [(3)H]acetylcholine ([(3)H]ACh) release by the A(2A) adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve-hemidiaphragm. The P-type calcium channel blocker omega-agatoxin IVA (100 nM) decreased [(3)H]ACh release evoked with pulses of 0.04-ms duration, whereas nifedipine (1 microM) inhibited transmitter release with pulses of 1-ms duration. Depletion of intracellular calcium stores by thapsigargin (2 microM) decreased [(3)H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04-ms) stimulation pulses, when P-type calcium influx triggered transmitter release, facilitation of [(3)H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 microM) and nifedipine (1 microM). With longer stimuli (1 ms), a situation in which both thapsigargin-sensitive internal stores and L-type channels are involved in ACh release, pretreatment with either omega-agatoxin IVA (100 nM) or nifedipine (1 microM) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A(2A) receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin-sensitive internal stores and/or P- or L-type channels) that are not committed to the release process in each stimulation condition.

Purinergic modulation of [ 3H]GABA release from rat hippocampal nerve terminals

The hippocampal GABAergic system is assumed not to be a target for purine modulation. We have now confirmed that neither adenosine A 1 and A 3 receptor nor nucleotide P 2 or P 4 receptor activation modified the K +-evoked [ 3H]GABA release from hippocampal synaptosomes. However, activation of adenosine A 2A receptors with CGS 21680 (10 nM) or HENECA (30 nM) facilitated GABA release by 32% and 21%, respectively. These effects were prevented by the A 2A antagonist, ZM 241385 (20 nM). A 2A receptors may activate adenylate cyclase and protein kinase A since CGS 21680 (10 nM) facilitation was partially prevented by 8-bromo-cAMP (1 mM), forskolin (10 μM) and HA-1004 (10 μM). Protein kinase C may also be recruited, since chelerythrine (6 μM) and phorbol-12,13-didecanoate (250 nM) attenuated CGS 21680 (10 nM) facilitation of [ 3H]GABA release. ω-Agatoxin-IVA (200 nM) occluded CGS 21680 facilitation suggesting the involvement of P-type calcium channels. Thus, the adenosine A 2A receptor system appears to be one of the first presynaptic neuromodulatory systems able to enhance the evoked release of GABA from hippocampal nerve terminals.

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Intrathecal drug therapy for chronic pain: from basic science to clinical practice.

Intrathecal drug therapy for chronic pain: from basic science to clinical practice.

The modulation of calcium current by GABA metabotropic receptors in a sub-population of pallidal neurons.

Globus pallidus (GP) receives an abundant GABAergic (gamma-aminobutyric acid) pathway from the corpus striatum. Several evidences suggested that alterations of this pathway might underlie the development of movement disorders. Classical models on Parkinsonism are centred on the increased excitability of GABAergic striatofugal neurons impinging GP and, therefore, on the presumed hypoactivity of GP neurons, but very few electrophysiological studies have addressed the activation of GABA receptors in mammalian GP. We have isolated calcium currents in GP neurons dissociated from the adult rat brain and analysed GABA-mediated responses. In the presence of bicuculline, the fast, chloride-mediated, ionotropic responses were obscured and GABA produced a large (>/= 35%) inhibition of calcium currents. The GABA-induced inhibition of calcium currents strongly desensitized was mimicked by baclofen and prevented by hydroxy-saclofen, supporting the involvement of GABAB receptors. The baclofen-mediated modulation was: (i) associated with slowing of activation kinetics; (ii) relieved by prepulse facilitation; and (iii) G-protein-mediated. The response was slow in onset, requiring the mobilization of intracellular cAMP, and was abolished by the combination of N-type and P-type calcium channel blockers. The GABAB-mediated effect, however, was confined to a particular subtype of GP neurons, identified by relatively small to medium soma. Differently, in cells characterized by larger somata and capacitance, the baclofen response was negligible. Intriguingly, these baclofen-resistant, larger neurons manifested a consistent low-voltage-activated (LVA) calcium current, not detected in baclofen-sensitive cells, at least when recorded in whole-cell mode. This study demonstrates that GP neurons express functional GABAA and GABAB receptors. In a subset of GP neurons, the activation of GABAB receptors induces a large modulation of high-voltage-activated (HVA) calcium currents, which may strongly influence basal ganglia circuitry and partially explain some discrepancies of classical models of extrapyramidal disorders.

Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to omega-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescence-labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

Effects of omega-agatoxin IVA, a P-type calcium channel antagonist, on the development of spinal neuronal hyperexcitability caused by knee inflammation in rats.

Both N- and P-type high-threshold calcium channels are located presynaptically in the CNS and are involved in the release of transmitters. To investigate the importance of P-type calcium channels in the generation of inflammation-evoked hyperexcitability of spinal cord neurons, electrophysiological recordings were made from wide-dynamic-range neurons with input from the knee joint in the anesthetized rat. The responses of each neuron to innocuous and noxious pressure onto the knee and the ankle were continuously assessed before and during the development of an inflammation in the knee joint induced by the injections of K/C into the joint cavity. The specific antagonist at P-type calcium channels omega-agatoxin was administered into a 30-microl trough on the spinal cord surface above the recorded neuron. In most neurons the application of omega-agatoxin before induction of inflammation slightly enhanced the responses to pressure onto the knee and ankle or left them unchanged. Two different protocols were then followed. In the control group (13 rats) only Tyrode was administered to the spinal cord during and after induction of inflammation. In these neurons the responses to mechanical stimuli applied to both the inflamed knee and to the noninflamed ankle showed a significant increase over 4 h. In the experimental group (12 rats) omega-agatoxin was applied during knee injection and in five 15-min periods up to 180 min after kaolin. This prevented the increase of the neuronal responses to innocuous pressure onto the knee and to innocuous and noxious pressure onto the ankle; only the responses to noxious pressure onto the knee were significantly enhanced during development of inflammation. Thus the development of inflammation-evoked hyperexcitability was attenuated by omega-agatoxin, and this suggests that P-type calcium channels in the spinal cord are involved in the generation of inflammation-evoked hyperexcitability of spinal cord neurons. Finally, when omega-agatoxin was administered to the spinal cord 4 h after the kaolin injection, i.e., when inflammation-evoked hyperexcitability was fully established, the responses to innocuous and noxious pressure onto the knee were reduced by 20-30% on average. The shift in the effect of omega-agatoxin, from slight facilitation or no change of the responses before inflammation to inhibition in the state of hyperexcitability, indicates that P-type calcium channels are important for excitatory synaptic transmission involved in the maintenance of inflammation-evoked hyperexcitability.

Calcium influx is required for endocytotic membrane retrieval.

Cells use endocytotic membrane retrieval to compensate for excess surface membrane after exocytosis. Retrieval is thought to be calcium-dependent, but the source of this calcium is not known. We found that, in sea urchin eggs, endocytotic membrane retrieval required extracellular calcium. Inhibitors of P-type calcium channels-cadmium, omega-conotoxin MVIIC, omega-agatoxin IVA, and omega-agatoxin TK-blocked membrane retrieval; selective inhibitors of N-type and L-type channels did not. Treatment with calcium ionophores overcame agatoxin inhibition in a calcium-dependent manner. Cadmium blocked membrane retrieval when applied during the first 5 minutes after fertilization, the period when the membrane potential is depolarized. We conclude that calcium influx through omega-agatoxin-sensitive channels plays a key role in signaling for endocytotic membrane retrieval.

Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons.

The antihypertensive agent mibefradil completely and reversibly inhibited T-type calcium channels in freshly isolated rat cerebellar Purkinje neurons. The potency of mibefradil was increased at less hyperpolarized holding potentials, and the apparent affinity was correlated with the degree of channel inactivation. At 35 degrees, the apparent dissociation constant Kapp was 1 microM at a holding voltage of -110 mV (corresponding to noninactivated channels) and 83 nM at a holding voltage of -70 mV (corresponding to 65% inactivation). The increased affinity was attributable mainly to a decreased off-rate. Mibefradil also inhibited P-type calcium channels in Purkinje neurons, but inhibition was much less potent. At a holding potential of -70 mV, the Kapp for mibefradil inhibition of P-type channels was approximately 200-fold higher than that for inhibition of T-type channels. Mibefradil should be a useful compound for distinguishing T-type channels from high voltage-activated calcium channels in neurons studied in vitro.

Single Tottering Mutations Responsible for the Neuropathic Phenotype of the P-type Calcium Channel

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.

Whole-Cell and Single-Channel Analysis of P-Type Calcium Currents in Cerebellar Purkinje Cells of Leaner Mutant Mice

The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study we used both whole-cell and single-channel patch-clamp recordings to examine the functional consequences of the tgla mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, we observed a marked reduction of peak current density in tgla/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells was accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents were predominantly of the omega-agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype.

Endothelin-stimulated capacitative calcium entry in enteric glial cells: synergistic effects of protein kinase C activity and nitric oxide.

Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 nM) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni2+ (89 +/- 2%) and by La3+ (78 +/- 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor NG-nitro-L-arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and NG-nitro-L-arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.