Inward Rectification (Ih) in Immunocytochemically-ldentified Vasopressin and Oxytocin Neurons of Guinea-Pig Supraoptic Nucleus

Intracellular recordings of magnocellular neurons from the supraoptic nucleus of guinea-pigs were made with KCI/K citrate- and biocytin-filled electrodes. Fifty of 99 cells exhibited a time-dependent inward rectification (TDR). The TDR was activated during hyperpolarizing current pulses to membrane potentials more hyperpolarized than −75 mV. In voltage-clamp recordings, an inward current appeared at voltage steps more hyperpolarized than −75 mV, with properties similar to the slow inward rectifier (I_h) described in other tissues. The I_h was blocked by 2 mM CsCI. BaCI₂ (100 to 500 μM) did not block the I_h. Immunocytochemical identification of the recorded cells revealed that both vasopressin (AVP)- and oxytocin (OT)- containing neurons exhibited an I_h.     

Widespread expression of olfactory cyclic nucleotide-gated channel genes in rat brain: implications for neuronal signalling

The cyclic nucleotides cAMP and cGMP are important intracellular messengers involved in a wide variety of signal transduction events in the nervous system. It has been proposed that cAMP/cGMP elicit some of their effects through direct gating of a novel class of Ca2+ -permeable ion channels that are termed cyclic nucleotide-gated (CNG) channels. Previous studies have identified the expression of a gene encoding one major CNG channel subtype, the olfactory receptor neuron alpha subunit, in the brain [El-Husseini et al. (1995) NeuroReport 6:1331-1335; Kingston et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93:10440-10445; Bradley et al. (1997) J. Neurosci. 17:1993-2085]. We, therefore, proposed that the actions of cAMP/cGMP on neurons in the brain might occur through the activation of these CNG channels. To determine how widespread such a function might be, the regional and cellular distribution of the olfactory CNG channel alpha subunit has been examined in detail. Primers for multiple portions of the olfactory CNG channel were used in polymerase chain reaction (PCR) to amplify cDNA reverse-transcribed from several brain regions. The identities of PCR products were confirmed with Southern blots and by sequencing. In situ hybridization experiments demonstrated localization of CNG channel mRNA in discrete neuronal populations throughout the brain. In agreement with previous work, relatively strong hybridization signals are present in neuronal cell bodies of the cerebellum, olfactory bulb, cerebral cortex, and brainstem. Additionally, somewhat lesser signals are found in thalamus, hypothalamus, midbrain, and spinal cord while no hybridization signal was detectable in the caudate nucleus. This surprisingly wide distribution throughout the rat brain strengthens the hypothesis that CNG channels may influence numerous processes as downstream effectors of cyclic nucleotide cascades. Interestingly, the distribution of CNG channels is very similar to that of the nitric oxide/cGMP system, suggesting that one function of CNG channels in the brain could be to link diffusible messengers to elevated Ca2+ entry into neurons.     

Excitability of pontine startle processing neurones is regulated by the two-pore-domain K+ channel TASK-3 coupled to 5-HT2C receptors

The mammalian startle reflex is a fast response to sudden intense sensory stimuli that can be increased by anxiety or decreased by reward. The cellular integration of sensory and modulatory information takes place in giant neurones of the caudal pontine reticular formation (PnC). The startle reflex is known to be enhanced by 5-hydroxytryptamine (5-HT); however, signalling mechanisms that change the excitability of the PnC giant neurones are poorly understood. Possible molecular candidates are two-pore-domain K⁺ (K₂P) channels that generate a variable K⁺ background conductance and control neuronal excitability upon activation of G-protein-coupled receptors. We demonstrate by in situ hybridization that the K₂P channel TASK-3 is substantially expressed in PnC giant neurones. Brain slice recordings revealed a corresponding background K⁺ current in these cells that forms about 30% of the outward current at −30 mV. Inactivation of TASK-3 at pH 6.4 and by ruthenium red depolarized the cells by about 7 mV and increased the action potential frequency as well as duration. Specific activation of Gα_q-coupled 5-HT₂ receptors with α-methyl 5-HT evoked a similar increase of neuronal excitability. Consistently, we measured afferent synaptic inputs from serotonergic raphe neurones and detected 5-HT_2C receptors in PnC giant neurones by immunohistochemistry. Thus, neuronal excitability of PnC giant neurones in vivo is most likely increased by serotonergic projections via the K₂P channel TASK-3.     

Voltage-dependent Currents in Microvillar Receptor Cells of the Frog Vomeronasal Organ

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 μm) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was −61 ± 13 mV (mean ± SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of –60 to –86 mV, with almost no spontaneous action potential. The input resistance was in the GΩ range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2 – 10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near –45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near –75 mV. An outward K⁺ current was blocked by internal Cs⁺ ions or by external tetraethylammonium or Ba²⁺ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd²⁺ ions. A voltage-dependent Ca²⁺ current was observed in an iso-osmotic BaCl₂ solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.     

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca²⁺]_c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na⁺ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K⁺ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca²⁺]_c, suggesting that prolongation of the duration of depolarization is required for somatic Ca²⁺ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca²⁺]_c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca²⁺ channels contribute to the field-evoked neurite response.     

Phosphorylation of voltage-gated ion channels in rat olfactory receptor neurons

In olfactory receptor neurons (ORNs), ligand-odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation.     

Comparative Patch-clamp Studies with Freshly Dissociated Rat Hippocampal and Striatal Neurons on the NMDA Receptor Antagonistic Effects of Amantadine and Memantine

Patch- and concentration-clamp techniques were used to compare the effects of the uncompetitive N -methyl-D-aspartate (NMDA) receptor antagonists (+)-MK-801 (dizocilpine, (+)-5-methyl-10, 11-dihydro-5H-dibenzo-cyclohepten-5, 10-imine maleate), ketamine, memantine (1-amino-3, 5-dimethyladamantane) and amantadine (1-amino-adamantane) on agonist-induced inward currents in freshly dissociated rat hippocampal and striatal neurons. In hippocampal neurons, ketamine (5 μM), memantine (10 μM) and amantadine (100 μM) selectively antagonized inward current responses to NMDA (500 μM plus glycine 5 μM) in a voltage-dependent manner without affecting responses to ( s )-α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (100 μM) or γ-aminobutyric acid (10 μM). The NMDA receptor antagonistic effect of all four agents was typical of open channel blockade. The kinetics of blockade/unblockade was inversely related to antagonist affinity. In hippocampal neurons amantadine was the least potent NMDA receptor antagonist (IC₅₀ 18.6 ± 0.9 μM) and showed the fastest blocking kinetics, whereas (+)-MK-801 was the most potent (IC₅₀ 0.12 ± 0.01 μM) and showed the slowest blocking kinetics. Memantine (IC₅₀ 1.04 ± 0.26 μM) and ketamine (IC₅₀ 0.43 ± 0.10 μM) were almost equipotent and had similar, intermediate blocking kinetics. In striatal neurons recorded under identical conditions (+)-MK-801, ketamine and memantine were 3- to 4-fold less potent whereas amantadine was somewhat more potent than on hippocampal neurons. This could offer an explanation for the better clinical profile of amantadine in Parkinson's disease, as therapeutically relevant concentrations of amantadine are likely to be more active in the striatum whereas memantine is likely to be more active in other structures.     

The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium.

Cation channels that are directly gated by guanosine 3', 5'-cyclic monophosphate (cGMP) control the flow of ions across the surface membrane of vertebrate rod and cone photoreceptor cells. A similar channel, gated by adenosine 3',5'-cyclic monophosphate (cAMP), exists in vertebrate olfactory sensory neurons. The channel polypeptide of rod photoreceptors has been identified and the amino acid sequence of the channel polypeptide in rod and olfactory cells has been determined by cloning cDNA. Although the cyclic nucleotide-gated channels functionally belong to the class of ligand-gated channels, they share some structural features with voltage-gated channels.     

Currents Activated by GABA and their Modulation by Zn2+ in Cerebellar Granule Cells in Culture

Whole-cell and single-channel currents evoked by γ-aminobutyric acid (GABA) were recorded from rat cerebellar granule cells in culture. The electro-physiological properties of these currents were studied in control condition and in the presence of external Zn²⁺ (10 – 30μM). GABA (10 μM) induced bicuculline-sensitive whole-cell currents which desensitized. The desensitization was more rapid for higher concentrations of GABA (30 – 300 μM). The current – voltage relation of GABA currents was linear from – 70 to +50 mV. Two different types of cells were found with respect to the stoichiometry for agonist binding, one with Hill coefficient 1.5 and another one with coefficient 1. The half-maximum concentration displayed more variability, with values varying from 10 to 50 μM. The time constant of recovery from desensitization (T_r) was estimated to be 36 s. Zn²⁺ (30 μM) blocked GABA-activated whole-cell currents in a non-competitive and voltage-independent way without a significant change in the current kinetics. In excised outside-out patches, GABA (0.5 μM) activated single-channel events of 19 and 31 pS. Kinetic analysis yielded two mean shut times (T_c1= 2.70 ms, T_c2= 205 ms) and one mean open time (T_o= 3.64 ms). Zn²⁺ (10 μM) did not affect single-channel conductances and mean open and shut times, but significantly reduced the probability of opening from 0.17 to 0.06. It is probable that Zn²⁺ binds to a site located on the extracellular part of the GABA_A receptor channel complex.     

Highly specific responses to amine odorants of individual olfactory receptor neurons in situ

The main olfactory system of larval Xenopus laevis is made up of at least two subsystems consisting of subsets of olfactory receptor neurons (ORNs) with different transduction mechanisms. One ORN subset lacks the canonical cAMP transduction pathway and responds to amino acid odorants. The second subset has the cAMP transduction pathway but as yet suitable odorants are unknown. Here we report the identification of amines as proper olfactory stimuli for larval X. laevis using functional Ca²⁺ imaging and slice preparations of the olfactory system. The response profiles of individual ORNs to a number of amines were extremely complex and mostly highly specific. The great majority of amine-sensitive ORNs responded also to forskolin, an activator of the olfactory cAMP transduction pathway. Most amine-induced responses could be attenuated by the cyclic nucleotide-gated channel inhibitor LY83583. This confirms that most amine-responsive olfactory receptors (ORs) are coupled to the cAMP-dependent transduction pathway. Furthermore, we show that trace amine-associated receptors (TAARs), which have been shown to act as specific ORs for amines in mammals, are expressed in the olfactory organ of X. laevis . The TAARs expressed in Xenopus cannot, however, explain the complex responses of individual ORNs to amines because there are too few of them. This indicates that, in addition to TAARs, there must be other receptor families involved in the detection of amines.     

Effects of a Cognition-enhancer, Linopirdine (DuP 996), on M-type Potassium Currents (IK(M)) Some Other Voltage- and Ligand-gated Membrane Currents in Rat Sympathetic Neurons

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K⁺ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 μM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (I_K(v)), IC50 63 μM; transient (I_A) current, IC50 69 μM] and produced no detectable inhibition of the fast and slow Ca²⁺-activated K⁺ currents I_c and I_AHP or of a hyperpolarization-activated cation current (I_Q/I_h), at 10–30 μM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl^- currents with IC50 values of 7.6 and 26 μM respectively. It is concluded that linopirdine shows some 20–fold selectivity for M-channels among different K⁺ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.     

Lamotrigine Reduces Voltage-Gated Sodium Currents in Rat Central Neurons in Culture

Summary: Purpose : To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA⁺ channels in mammalian central neurons.
Methods : Rat cerebellar granule cells in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na⁺ currents.
Results : Extracellular application of LTG (10–500 μ M , n = 21) decreased in a dose-related manner a tetrodotoxin-sensitive inward current that was elicited by depolarizing commands (from −80 to +20mV). The peak amplitude of this Na⁺-mediated current was diminished by 38.8 ± 12.2% (mean ± SD, n = 6) during application of 100 μ M LTG, and the dose-response curve of this effect indicated an IC₅₀ 145 μM. The reduction in the inward currents produced by LTG was not associate with any signficant change in the current decay, whereas the voltage dependency of the steady-state inactivation shifted toward more negative values (midpoint of the inactivation curve: –47.5 and –59.0 mV under control conditions and during application of 100 μM LTG, respectively, n = 4).
Conclusions : Our findings indicate that LTG reduces the amplitude of voltage-gated Na⁺ inward current in rat cerebellar granule cells and induces a negative shift of the steady-state inactivation curve. Both mechanisms may be instrumental in controlling the repetitive firing of action potentials (AP) that occurs in neuronal networks during seizure activity.     

Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

Expression of olfactory-type cyclic nucleotide-gated channels in rat cortical astrocytes

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.     

Cable Properties of Dendrites in Hippocampal Neurons of the Rat Mapped by a Voltage-sensitive Dye

Dendrites of pyramidal neurons from embryonic rat hippocampus are investigated in culture using a voltage-sensitive fluorescent dye. The electrical response to somatic stimulation is observed as a time-resolved map with a resolution of 0.9 μM at a time constant of 0.4 ms without signal averaging. The data are interpreted in terms of a tapering cable with Hodgkin-Huxley parametrization. The spread of short hyperpolarizing transients is damped by capacitive shunting. The invasion of an action potential is boosted by voltage-gated conductances of a low density. No irregularity is observed at a bifurcation. The passive cable parameters of internal resistance and membrane resistance at resting voltage are R_i= 300 ωcm and R_m= 40 kωcm² respectively, at a maximum sodium conductance of ˜4.4 mS/cm². The electrotonic length constant and the dynamic length constant at 1 kHz are 580 and 90 μm respectively. These results are compatible with electrophysiological data of dendrites in slices of adult hippocampus and with optical data of narrow processes of leech neurons in culture. The functional implications of boosting an action potential by voltage-gated channels of low density are considered.     

Functional Heterogeneity of Hippocampal GABAA Receptors

γ-Aminobutyric acid type A (GABA_A) receptors were studied in cultured neurons taken from rat hippocampus at early postnatal stages. GABA-induced whole-cell currents showed a broad range of peak amplitudes and time-courses of desensitization. Dose – response curves of rapidly and slowly desensitizing cells revealed EC₅₀ values of 8.5 and 37.3 μM GABA, respectively, with the Hill coefficient being greater than unity. The main-state conductance of GABA_A receptor channels was 28 – 31 pS in all cells. GABA responses of low-affinity cells were more strongly affected by benzodiazepine receptor agonists (e.g. flunitrazepam, clonazepam) and inverse agonists (e.g. methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate), as compared to cells exhibiting high-affinity GABA responses. Currents were also potentiated by zolpidem, but were little affected by Ro 15-4513 and Zn²⁺. These data suggest the presence of physiologically and pharmacologically distinct GABA_A receptor isoforms in neurons of the early postnatal hippocampus, which may subserve different inhibitory control mechanisms in this brain region.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

Activation of Nucleotide Receptors Inhibits High-threshold Calcium Currents in NG108-15 Neuronal Hybrid Cells

A P_2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma × glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K⁺-current. We now report that UTP also inhibits Ca²⁺-currents in differentiated NG 108-15 cells, but probably through a different nucleotide receptor. UTP (100 μM) inhibited the peak of the high-threshold current by 28.4 ± 3.1% ( n = 38) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM ω-conotoxin (CgTx) and one inhibited by 2 μM nifedipine and enhanced by 1 μM BAY K8644. UTP inhibited the former by 31.0 ± 3.1%, with an IC₅₀ of 2.8 ± 1.1 μM, and the latter by 34.2 ± 6.1% with an IC₅₀ of 1.7 ± 1.3 μM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or CAMP (1 mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP₄A and ATPγS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca²⁺-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals.     

CNG-modulin: a novel Ca-dependent modulator of ligand sensitivity in cone photoreceptor cGMP-gated ion channels

The transduction current in several different types of sensory neurons arises from the activity of cyclic nucleotide-gated (CNG) ion channels. The channels in these sensory neurons vary in structure and function, yet each one demonstrates calcium-dependent modulation of ligand sensitivity mediated by the interaction of the channel with a soluble modulator protein. In cone photoreceptors, the molecular identity of the modulator protein was previously unknown. We report the discovery and characterization of CNG-modulin, a novel 301 aa protein that interacts with the N terminus of the β subunit of the cGMP-gated channel and modulates the cGMP sensitivity of the channels in cone photoreceptors of striped bass (Morone saxatilis). Immunohistochemistry and single-cell PCR demonstrate that CNG-modulin is expressed in cone but not rod photoreceptors. Adding purified recombinant CNG-modulin to cone membrane patches containing the native CNG channels shifts the midpoint of cGMP dependence from ～91 μM in the absence of Ca(2+) to ～332 μM in the presence of 20 μM Ca(2+). At a fixed cGMP concentration, the midpoint of the Ca(2+) dependence is ～857 nM Ca(2+). These restored physiological features are statistically indistinguishable from the effects of the endogenous modulator. CNG-modulin binds Ca(2+) with a concentration dependence that matches the calcium dependence of channel modulation. We conclude that CNG-modulin is the authentic Ca(2+)-dependent modulator of cone CNG channel ligand sensitivity. CNG-modulin is expressed in other tissues, such as brain, olfactory epithelium, and the inner ear, and may modulate the function of ion channels in those tissues as well.     

Adenosine Inhibits L- and N-Type Calcium Channels in Pituitary Melanotrophs. Evidence for the Involvement of a G Protein in Calcium Channel Gating

It has been previously demonstrated that activation of A₁ adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A₁ adenosine receptor agonist R-PIA (50 μM each) produced a decrease of the amplitude of the barium current, while the selective A₂ adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTPyS (100 μM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 μg/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 μM) together with the phosphodiesterase inhibitor IBMX (100 μM) to the intracellular solution did not modify the effect of R-PIA on the current.
It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.