Voltage-independent Inhibition Research Articles

Unique Isoform of Gα-interacting Protein (RGS-GAIP) Selectively Discriminates between Two Go-mediated Pathways That Inhibit Ca2+ Channels

Regulators of G-protein signaling (RGS) proteins constitute a large family of GTPase-activating proteins for heterotrimeric G proteins. More than 20 RGS genes have been identified in mammals. One of these, the Galpha-interacting protein (GAIP), preferentially interacts with members of the G(i)/G(o) subfamily of G proteins in mammalian cells, but its selectivity among members of this subfamily in vitro is limited. Here we report the cloning and functional characterization of a unique cDNA isoform of GAIP, derived from embryonic chicken dorsal root ganglion neurons. Chick GAIP is composed of 199 amino acids, organized into a conserved RGS domain (85% identical to human GAIP), and a unique, short N terminus (only 41% identical, 50% homologous to known mammalian orthologues). Consistent with this unique primary structure, chick GAIP has physiological properties that distinguish it from mammalian GAIPs. We have explored the selectivity of chick GAIP in electrophysiological assays of two G(o)-mediated forms of Ca(2+) channel inhibition produced by gamma-aminobutyric acid in chick dorsal root ganglion neurons, voltage-independent inhibition (mediated by G(o)alpha) and voltage-dependent inhibition (mediated by G(o)betagamma). Dialyzing recombinant chick GAIP in these cells selectively reduced voltage-independent inhibition without affecting voltage-dependent inhibition. Mammalian GAIP, tested under identical conditions in previous studies, demonstrated no selectivity between these two inhibitory processes; thus, our results suggest that the functional specificity of chick GAIP is likely to be determined by its unique N terminus.

The role of histidine residues in modulation of the rat P2X(2) purinoceptor by zinc and pH.

P2X(2) receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC(50) of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 microM ATP of wild-type P2X(2) and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pK(a) (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X(2) and revealed a substantial pH-dependent decrease in the maximum response with a pK(a) near 6.0. Substituting a lysine for H319 reduced the EC(50) for ATP 40-fold. Zinc (20 microM) potentiated the responses to 10 microM ATP of wild-type P2X(2) and seven histidine mutants by approximately 8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

Inhibition of acetylcholine-activated K(+) currents by U73122 is mediated by the inhibition of PIP(2)-channel interaction.

1. We have investigated the effect of U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine-activated K(+) currents (I(KACh)) in mouse atrial myocytes. 2. In perforated patch clamp mode, I(KACh) was activated by 10 microM acetylcholine. When atrial myocytes were pretreated with U73122 or U73343, I(KACh) was inhibited dose-dependently (half-maximal inhibition at 0.12+/-0.0085 and 0.16+/-0.0176 microM, respectively). The current-voltage relationships for I(KACh) in the absence and in the presence of U73122 showed that the inhibition occurred uniformly from -120 to +40 mV, indicating a voltage-independent inhibition. 3. When U73122 was applied after I(KACh) reached steady-state, a gradual decrease in I(KACh) was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of U73122. 4. When K(ACh) channels were directly activated by adding 1 mM GTP gamma S to the bath solution in inside-out patches, U73122 (1 microM) decreased the open probability significantly without change in mean open time. When K(ACh) channels were activated independently of G-protein activation by 20 mM Na(+), open probability was also inhibited by U73122. 5. Voltage-activated K(+) currents and inward rectifying K(+) currents were not affected by U73122. 6. These findings show that inhibition by U73122 and U73343 of K(ACh) channels occurs at a level downstream of the action of G beta gamma or Na(+) on channel activation. The interference with phosphatidylinositol 4,5-bisphosphate (PIP(2))-channel interaction can be suggested as a most plausible mechanism.

Voltage-independent Inhibition of P/Q-type Ca2+Channels in Adrenal Chromaffin Cells via a Neuronal Ca2+Sensor-1-dependent Pathway Involves Src Family Tyrosine Kinase

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.

Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel.

Gamma-aminobutyric acid (GABA)B receptors couple to Go to inhibit N-type calcium channels in embryonic chick dorsal root ganglion neurons. The voltage-independent inhibition, mediated by means of a tyrosine-kinase pathway, is transient and lasts up to 100 seconds. Inhibition of endogenous RGS12, a member of the family of regulators of G-protein signalling, selectively alters the time course of voltage-independent inhibition. The RGS12 protein, in addition to the RGS domain, contains PDZ and PTB domains. Fusion proteins containing the PTB domain of RGS12 alter the rate of termination of the GABA(B) signal, whereas the PDZ or RGS domains of RGS 12 have no observable effects. Using primary dorsal root ganglion neurons in culture, here we show an endogenous agonist-induced tyrosine-kinase-dependent complex of RGS12 and the calcium channel. These results indicate that RGS12 is a multifunctional protein capable of direct interactions through its PTB domain with the tyrosine-phosphorylated calcium channel. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways.

Ca2+-dependent desensitization of AMPA receptors.

The effect of changes in the external concentrations (0.4-10 mM) of Ca2+ ions on AMPA receptors (AMPARs) of different subunit composition was studied on freshly isolated rat brain neurones. Ca2+ produces rapid and reversible voltage-independent inhibition of AMPARs. Ca2+-permeable and Ca2+-impermeable AMPARs are equally sensitive to external Ca2+ suggesting that the effect is not addressed to the ion channel. The inhibition of responses evoked by AMPA is significantly larger than those evoked by kainate or glutamate. Cyclothiazide and aniracetam, which are known to prevent AMPAR desensitization, both greatly diminish inhibition of AMPARs by Ca2+. Cyclothiazide is more potent than aniracetam in both preventing of AMPAR desensitization and protecting against the Ca2+ inhibitory effect on hippocampal pyramidal cells. On giant cholinergic interneurones of striatum, aniracetam but not cyclothiazide significantly prevents inhibition by Ca2+. This agrees with available data on relative abundance of flip and flop splice variants in these cell types. The results suggest that Ca2+ may allosterically increase AMPA receptor desensitization independently on subunit composition and splice variants.

Src Potentiation of NMDA Receptors in Hippocampal and Spinal Neurons Is Not Mediated by Reducing Zinc Inhibition

The protein-tyrosine kinase Src is known to potentiate the function of NMDA receptors, which is necessary for the induction of long-term potentiation in the hippocampus. With recombinant receptors composed of NR1-1a/NR2A or NR1-1a/2B subunits, Src reduces voltage-independent inhibition by the divalent cation Zn2+. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ level is sufficient to cause tonic inhibition. Here we investigated whether the Src-induced potentiation of NMDA receptor function in neurons is caused by reducing voltage-independent Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked the Src-induced potentiation of NR1-1a/2A receptors, we found that Zn2+ chelation did not affect the potentiation of NMDA receptor (NMDAR) currents by Src applied into hippocampal CA1 or CA3 neurons. Moreover, Src did not alter the Zn2+ concentration-inhibition relationship for NMDAR currents in CA1 or CA3 neurons. Also, chelating extracellular Zn2+ did not prevent the upregulation of NMDA single-channel activity by endogenous Src in membrane patches from spinal dorsal horn neurons. Taking these results together we conclude that Src-induced potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.

G-Proteins Are Involved in 5-HT Receptor-Mediated Modulation of N- and P/Q- But Not T-Type Ca2+Channels

5-HT produces voltage-independent inhibition of the N-, P/Q-, and T-type Ca2+ currents in sensory neurons of Xenopus larvae by acting on 5-HT1A and 5-HT1D receptors. We have explored the underlying mechanisms further and found that the inhibition of high voltage-activated (HVA) currents by 5-HT is mediated by a pertussis toxin-sensitive G-protein that activates a diffusible second messenger. Although modulation of T-type currents is membrane-delimited, it was not affected by GDP-beta-S (2 mM), GTP-gamma-S (200 microM), 5'-guanylyl-imidodiphosphate tetralithium (200 microM), aluminum fluoride (AlF4-, 100 microM), or pertussis toxin, suggesting that a GTP-insensitive pathway was involved. To investigate the modulation of the T currents further, we synthesized peptides that were derived from conserved cytoplasmic regions of the rat 5-HT1A and 5-HT1D receptors. Although two peptides derived from the third cytoplasmic loop inhibited the HVA currents by activating G-proteins and occluded the modulation of HVA currents by 5-HT, two peptides from the second cytoplasmic loop and the C tail had no effect. None of the four receptor-derived peptides had any effect on the T-type currents. We conclude that 5-HT modulates T-type channels by a membrane-delimited pathway that does not involve G-proteins and is mediated by a functional domain of the receptor that is distinct from that which couples to G-proteins.

Divalent cation permeability and blockade of Ca2+-permeant non-selective cation channels in rat adrenal zona glomerulosa cells.

1. The effects of the divalent cations Ca2+, Mg2+ and Ni2+ on unitary Na+ currents through receptor-regulated non-selective cation channels were studied in inside-out and cell-attached patches from rat adrenal zona glomerulosa cells. 2. External Ca2+ caused a concentration-dependent and voltage-independent inhibition of inward Na+ current, exhibiting an IC50 of 1.4 mM. The channel was also Ca2+ permeant and external Ca2+ shifted the reversal potential as expected for a channel exhibiting a constant Ca2+ : Na+ permeability ratio near to 4. 3. External and internal 2 mM Mg2+ caused voltage-dependent inhibition of inward and outward Na+ current, respectively. Modelling Mg2+ as an impermeant fast open channel blocker indicated that external Mg2+ blocked the pore at a single site exhibiting a zero voltage Kd of 5.1 mM for Mg2+ and located 19 % of the distance through the transmembrane electric field from the external surface. Internal Mg2+ blocked the pore at a second site exhibiting a Kd of 1.7 mM for Mg2+ and located 36% of the distance through the transmembrane electric field from the cytosolic surface. 4. External Ni2+ caused a voltage- and concentration-dependent slow blockade of inward Na+ current. Modelling Ni2+ as an impermeant slow open channel blocker indicated that Ni2+ blocked the pore at a single site exhibiting a Kd of 1.09 mM for Ni2+ and located 13.7% of the distance through the transmembrane electric field from the external surface. 5. External 2 mM Mg2+ increased the Kd for external Ni2+ binding to 1.27 mM, consistent with competition for a single binding site. Changing ionic strength did not substantially affect Ni2+ blockade indicating the absence of surface potential under physiological ionic conditions. 6. It is concluded that at least two divalent cation binding sites, separated by a high free energy barrier (the selectivity filter), are located in the pore and contribute to Ca2+ selectivity and permeability of the channel.

Insensitivity of volume-sensitive chloride currents to chromones in human airway epithelial cells.

Chromones (sodium cromoglycate and sodium nedocromil) block cell swelling-activated Cl- channels in NIH-3T3 fibroblasts and endothelial cells. This has led to hypothesize that cell volume regulation might be involved in asthma pathogenesis. Using whole-cell patch-clamp experiments, we studied the effect of chromones on volume-sensitive Cl- currents in transformed human tracheal epithelial cells (9HTEo-) and in primary cultures of human bronchial epithelial cells (BE). Cl- currents activated by hypotonic shock were poorly blocked by extracellular nedocromil or cromoglycate. The block was voltage-dependent since it was observed only at positive membrane potentials. At the concentration of 5 mM, the current inhibition by both chromones at +80 mV was about 40% for 9HTEo- and only 20% for BE. Intracellular application of chromones elicited a voltage-independent inhibition in 9HTEo- cells. Under this condition, volume-sensitive Cl- currents were reduced at all membrane potentials (60 and 45% inhibition by 2 mM nedocromil and cromoglycate respectively). In contrast intracellular chromones were ineffective in BE cells. The relative refractoriness to chromones, in contrast with the high sensitivity shown by other Cl- channels, suggests that the epithelial volume-sensitive Cl- channel is not involved in asthma.

Glibenclamide blocks volume-sensitive Cl- channels by dual mechanisms.

To study the mechanisms of glibenclamide actions on volume-sensitive Cl- channels, whole cell patch-clamp studies were performed at various pH levels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl- currents in the entire range of voltage examined (-100 to +100 mV) and accelerated the depolarization-induced inactivation at pH 7.5. When glibenclamide was applied from the intracellular side, in contrast, no effect was observed. At acidic pH, at which the weak acid glibenclamide exists largely in the uncharged form, the instantaneous current was, in a voltage-independent manner, suppressed by the extracellular drug at micromolar concentrations without significantly affecting the depolarization-induced inactivation. At alkaline pH, at which almost all of the drug is in the charged form, glibenclamide speeded the inactivation time course and induced a leftward shift of the steady-state inactivation curve at much higher concentrations. Thus it is concluded that glibenclamide exerts inhibiting actions on swelling-activated Cl- channels from the extracellular side and that the uncharged form is mainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.

Differential inhibition of N and P/Q Ca2+ currents by 5-HT1A and 5-HT1D receptors in spinal neurons of Xenopus larvae.

1. In whole-cell patch clamp recordings made from non-sensory neurons acutely isolated from the spinal cord of Xenopus (stage 40-42) larvae, two forms of inhibition of the high voltage-activated (HVA) Ca2+ currents were produced by 5-HT. One was voltage dependent and associated with both slowing of the activation kinetics and shifting of the voltage dependence of the HVA currents. This inhibition was relieved by strong depolarizing prepulses. A second form of inhibition was neither associated with slowing of the activation kinetics nor relieved by depolarizing prepulses and was thus voltage independent. 2. In all neurons examined, 5-HT (1 microM) reversibly reduced 34 +/- 1.6 % (n = 102) of the HVA Ca2+ currents. In about 40 % of neurons, the inhibition was totally voltage independent. In another 5 %, the inhibition was totally voltage dependent. In the remaining neurons, inhibition was only partially (by around 40 %) relieved by a large depolarizing prepulse, suggesting that in these, the inhibition consisted of both voltage-dependent and -independent components. 3. By using selective channel blockers, we found that 5-HT acted on both N- and P/Q-type channels. However, whereas the inhibition of P/Q-type currents was only voltage independent, the inhibition of N-type currents had both voltage-dependent and -independent components. 4. The effects of 5-HT on HVA Ca2+ currents were mediated by 5-HT1A and 5-HT1D receptors. The 5-HT1A receptors not only preferentially caused voltage-independent inhibition, but did so by acting mainly on the omega-agatoxin-IVA-sensitive Ca2+ channels. In contrast, the 5-HT1D receptor produced both voltage-dependent and -independent inhibition and was preferentially coupled to omega-conotoxin-GVIA sensitive channels. This complexity of modulation may allow fine tuning of transmitter release and calcium signalling in the spinal circuitry of Xenopus larvae.

G-proteins and G-protein subunits mediating cholinergic inhibition of N-type calcium currents in sympathetic neurons.

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca2+ currents: this reduces Ca2+-activated K+ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M1 mAChRs [Hille (1994) Trends in Neurosci., 17, 531-536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their alpha and betagamma subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, overexpression of selected constitutively active subunits, and antagonism of endogenously liberated betagamma subunits by over-expression of Dy-binding P-adrenergic receptor kinase 1 (PARK1) peptide. The results indicate that: (i) M4 mAChR-induced inhibition is mediated by GoA; (ii) a and Py subunits released from the activated GoA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M1 mAChR-induced inhibition is likely to be mediated by the alpha subunit of Gq. Hence, Ca2+ current inhibition results from the concerted, but independent actions of three different G-protein subunits.

Sensitivity to Voltage-Independent Inhibition Determined by Pore-Lining Region of the Acetylcholine Receptor

Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). This study characterizes the mechanism of selective long-term inhibition of neuronal and muscle-neuronal chimeric nAChRs by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (bis-TMP-10 or BTMPS), a bifunctional form of the potent ganglionic blocker tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been previously demonstrated to arise, at least in part, from the binding of the bis compound to neuronal β-subunits. In this study, long-term inhibition is demonstrated to be dependent upon the presence of sequence element(s) within the pore-lining second transmembrane domain (tm2) of neuronal β-subunits; however, the inhibitor binding site itself does not appear to be contained within the segment of the channel pore influenced by the membrane electric field. Specifically, our results imply that bis-TMP-10 interacts with an activation-sensitive element, the availability of which may be regulated by a sequence in the tm2 domain. Furthermore, we demonstrate a compound length requirement for long-term inhibition that would be consistent with binding to multiple sites located on the extracellular portion of the receptor.

On the role of endogenous G-protein beta gamma subunits in N-type Ca2+ current inhibition by neurotransmitters in rat sympathetic neurones.

1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.

An active-site histidine of NR1/2C mediates voltage-independent inhibition by zinc

Endogenous zinc is an important modulator of ion channels of the central nervous system. To understand mechanisms of zinc inhibition, cloned heteromeric N-methyl- d-aspartate receptors (primary subunit NR1 with secondary subunits NR2A, NR2C or NR2D) were expressed in Xenopus oocytes and studied under two-electrode voltage-clamp. Voltage-independent inhibition of NR1/2A heteromers by nanomolar concentrations of extracellular zinc was observed in barium-containing perfusion solutions. In contrast, voltage-independent zinc inhibition of NR1/2C heteromers occurred with lower affinity. Zinc inhibition data from NR1/2D heteromers was fit well with a voltage-independent one-site model and resembled that previously reported for NR1/2B. Reduction of zinc inhibition of NR1/2C heteromers was seen after labeling with the histidine-modifying reagent diethylpyrocarbonate. This finding suggests that the NR1/2C heteromeric ion channel contains an active-site histidine responsible for zinc inhibition.

High-Affinity Zinc Inhibition of NMDA NR1–NR2A Receptors

Micromolar concentrations of extracellular Zn2+ are known to antagonize native NMDA receptors via a dual mechanism involving both a voltage-independent and a voltage-dependent inhibition. We have tried to evaluate the relative importance of these two effects and their subunit specificity on recombinant NMDA receptors expressed in HEK 293 cells and Xenopus oocytes. The comparison of NR1a-NR2A and NR1a-NR2B receptors shows that the voltage-dependent inhibition is similar in both types of receptors but that the voltage-independent inhibition occurs at much lower Zn2+ concentrations in NR1a-NR2A receptors (IC50 in the nanomolar range) than in NR1a-NR2B receptors (IC50 in the micromolar range). The high affinity of the effect observed with NR1a-NR2A receptors was found to be attributable mostly to the slow dissociation of Zn2+ from its binding site. By analyzing the effects of Zn2+ on varied combinations of NR1 (NR1a or NR1b) and NR2 (NR2A, NR2B, NR2C), we show that both the NR1 and the NR2 subunits contribute to the voltage-independent Zn2+ inhibition. We have observed further that under control conditions, i.e., in zero nominal Zn2+ solutions, the addition of low concentrations of heavy metal chelators markedly potentiates the responses of NR1a-NR2A receptors, but not of NR1a-NR2B receptors. This result suggests that traces of a heavy metal (probably Zn2+) contaminate standard solutions and tonically inhibit NR1a-NR2A receptors. Chelation of a contaminant metal also could account for the rapid NR2A subunit-specific potentiations produced by reducing compounds like DTT or glutathione.

Effect of englitazone on KATP and calcium-activated non-selective cation channels in CRI-G1 insulin-secreting cells.

1. The effects of englitazone sodium, an antidiabetic agent, on ion channel activity in the CRI-G1 insulin secreting cell line was examined by use of the patch clamp technique. 2. Application of englitazone to the outside of CRI-G1 cells in the whole-cell recording configuration produced concentration-dependent inhibition of KATP currents with an IC50 value of 8 microM. The inhibition of the K+ current was not affected by the removal of Mg2+ ions from or the addition of trypsin to the solution bathing the intracellular surface of the cell membrane. 3. Englitazone also inhibited KATP channel activity in recordings from inside out excise membrane patches. The concentration-dependence of inhibition was identical to that observed in whole-cell recordings and was voltage-independent. Single channel recordings confirmed that neither the absence or presence of Mg2+ ions nor the addition of trypsin at the intracellular surface of the membrane influenced the inhibition of KATP channels by englitazone. 4. Englitazone also inhibited Ca(2+)-activated non-selective cation (NSCa) channels in inside-out patches in a concentration-dependent and voltage-independent manner with an IC50 value of 10 microM. In comparison, the non-sulphonylurea KATP channel blocker ciclazindol produced a slight voltage-dependent inhibition of the NSCa channel at a concentration of 20 microM. 5. In whole-cell recordings englitazone, at a relatively high concentration (50 microM) in comparison with that required to block KATP and NSCa channels, inhibited voltage-activated Ca2+ currents by 33% but did not inhibit voltage-activated K+ and Na+ currents. 6. It is concluded that englitazone is a novel blocker of NSCa and KATP channels. The inhibition of KATP channels occurs following procedures that dissociate sulphonylurea receptor coupling to the channel. The equipotent and voltage-independent inhibition of NSCa and KATP channels by englitazone may indicate a common mechanism of block.

P-299 - Voltage-independent inhibition of cilnidipine on the ωconotoxin-sensitive component of the neuronal voltage dependent Ca2+-channel in rat

P-299 - Voltage-independent inhibition of cilnidipine on the ωconotoxin-sensitive component of the neuronal voltage dependent Ca2+-channel in rat

Opioid inhibition of Ca2+ channel subtypes in bovine chromaffin cells: selectivity of action and voltage-dependence.

Bovine chromaffin cells possess a mixture of high-voltage-activated Ca2+ channel subtypes: L-type, dihydropyridine-sensitive channels, and N-, P- and Q-types, omega-conotoxin MVIIC-sensitive channels. In these cells, we studied the reversible, naloxone-antagonized inhibition of Ba2+ currents by the opioid agonist met-enkephalin (IC50 = 272 nM). This inhibition could be resolved into a voltage-dependent and a voltage-independent component. The first was revealed by its slow Ba2+ current activation kinetics at 0 mV and by the current facilitation induced by short prepulses to +90 mV. The second was estimated as the residual inhibition persisting after the facilitation protocol. The two inhibitory components varied markedly from cell to cell and each contributed to about half of the total inhibition. Replacement of internal GTP by GDP-beta-S or cell pretreatment with pertussis toxin completely abolished the voltage-dependent inhibition by opioids, partially preserving the voltage-independent component. The opioid-induced inhibition was not selective for any Ca2+ channel subtype, being not prevented after the addition of specific Ca2+ channel antagonists. However, when separately analysing the contribution of each channel type to the voltage-dependent and voltage-independent modulation, a clear-cut distinction could be achieved. The voltage-independent inhibition was effective on all Ca2+ channel subtypes but predominantly on L-type Ca2+ channels. The voltage-dependent process was abolished by omega-conotoxin-MVIIC, but unaffected by nifedipine, and was thus sharply restricted to non-L-type channels (N-, P- and Q-types). Our data suggest a functionally distinct opioid receptor-mediated modulation of L- and non-L-type channels, i.e. of the two channel classes sharing major control of catecholamine secretion from bovine chromaffin cells.