Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition ... more Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the group III receptors mGluR4, mGluR6, and mGluR7. [3H]L-AP4 binding was present in human embryonic kidney cells transfected with the high-affinity mGluR4 receptor but not in cells transfected with mGluR6 or the low-affinity mGluR7 receptor. Analysis of mGluR4/mGluR6 receptor chimeras revealed that replacement of the first 35 amino acids of mGluR6 with the first 50 amino acids of mGluR4 was sufficient to impart [3H]L-AP4 binding to mGluR6. Homology models of mGluR4 and mGluR7 were used to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high-affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, whereas the N74K mutation in combination with mutations in residues 258 to 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared with unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the group III mGluRs.
The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain... more The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naı̈ve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.
Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of... more Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of nicotinic agonists improve attention, and nicotinic antagonist exposure impairs it. This study was conducted to investigate the effect of a novel nicotinic receptor agonist at α4β2 nicotinic receptors (AZD3480) on attention and reversal of pharmacologically induced attentional impairment produced by the NMDA glutamate antagonist dizocilpine (MK-801). Adult female Sprague-Dawley rats were trained to perform an operant visual signal detection task to a stable baseline of accuracy. The rats were then injected subcutaneously following a repeated measures, counter-balanced design with saline, AZD3480 (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations 30 min before the test. The effect of donepezil on the same pharmacologically induced attentional impairment was also tested. A separate group of rats was injected with donepezil (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations, and their attention were assessed. Saline was the vehicle control. Dizocilpine caused a significant (p < 0.0005) impairment in percent correct performance. This attentional impairment was significantly (p < 0.0005) reversed by 0.01 and 0.1 mg/kg of AZD3480. AZD3480 by itself did not alter the already high baseline control performance. Donepezil (0.01-1.0 mg/kg) also significantly (p < 0.005) attenuated the dizocilpine-induced attentional impairment. AZD3480, similar to donepezil, showed significant efficacy for counteracting the attentional impairment caused by the NMDA glutamate antagonist dizocilpine. Low doses of AZD3480 may provide therapeutic benefit for reversing attentional impairment in patients suffering from cognitive impairment due to glutamatergic dysregulation and likely other attentional disorders.
We have cloned two splice variants of the human homolog of the ␣ 1A subunit of voltage-gated Ca 2... more We have cloned two splice variants of the human homolog of the ␣ 1A subunit of voltage-gated Ca 2ϩ channels. The sequences of human ␣ 1A-1 and ␣ 1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human ␣ 1A-2 ␣ 2b ␦ 1b Ca 2ϩ channels expressed in HEK293 cells activate rapidly ( ϩ10mV ϭ 2.2 ms), deactivate rapidly ( -90mV ϭ 148 s), inactivate slowly ( ϩ10mV ϭ 690 ms), and have peak currents at a potential of ϩ10 mV with 15 mM Ba 2ϩ as charge carrier. In HEK293 cells transient expression of Ca 2ϩ channels containing ␣ 1A/B(f) , an ␣ 1A subunit containing a 112 amino acid segment of ␣ 1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba 2ϩ currents that were 30-fold larger compared to wild-type (wt) ␣ 1A-2containing Ca 2ϩ channels, and had inactivation kinetics similar to those of ␣ 1B-1 -containing Ca 2ϩ channels. Cells transiently transfected with ␣ 1A/B(f) ␣ 2b ␦ 1b expressed higher levels of the ␣ 1 , ␣ 2b ␦, and  1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N 1655 P 1656 ) and S5-SS1 (E 1740 ) that influence the biophysical properties of ␣ 1A . ␣ 1A E1740R resulted in a threefold increase in current magnitude, a Ϫ10 mV shift in steady-state inactivation, and an altered Ba 2ϩ current inactivation, but did not affect ion selectivity. The deletion mutant ␣ 1A ⌬NP shifted steady-state inactivation by Ϫ20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by -Aga IVA was increased with ␣ 1A ⌬NP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of ␣ 1A -containing Ca 2ϩ channels.
Electrophysiological investigations were carried out on several independently isolated mutants of... more Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1- 6 photoreceptors, and a mutant of theninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodop- sin content in R1-6 photoreceptors is reduced by 10 2-106-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologi- cally normal . Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model . Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps . Mutant bumpsare strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletio...
The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by... more The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by the second messenger, cyclic GMP. Injection of cGMP into rods or application of cGMP to inside-out membrane patches activates a conductance resembling that produced by light. Light produces a rapid reduction of cGMP in living rods, leading to closure of sodium channels and membrane hyperpolarization. In most invertebrate photoreceptors the response to light is depolarizing. We have investigated whether cGMP is involved in controlling the increase in sodium conductance that underlies this depolarization. We show here that injection of cGMP into Limulus photoreceptors produces a depolarization that mimics the receptor potential. We also show that the cGMP concentration of the squid retina increases rapidly during exposure to light. These results support the hypothesis that cGMP mediates the light-induced depolarization in invertebrate photoreceptors and suggests that vertebrate and invertebrate phototransduction may be more similar than previously thought.
In this review we have discussed the problem of deactivation at both the rhodopsin and G protein ... more In this review we have discussed the problem of deactivation at both the rhodopsin and G protein levels. Of particular interest is the novel observation that rhodopsin deactivation can be modulated by light. This modulation is likely to play an important role in light adaptation by reducing the gain of transduction. One interesting possibility is that this modulation involves the phosphorylation of an arrestin-like molecule, but this remains to be tested. One of the experimental advantages of Limulus photoreceptors is the large size of the single photon responses and the fact that even single G proteins produce a detectable response. This made possible the observation that nonhydrolyzable GTP analogues produce discrete transient events rather than the step-like events that would be predicted by previous models. This observation led us to a new view of how enzyme deactivation is coupled to GTP hydrolysis on G protein. According to this view, enzymes are activated by G protein, but ca...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1995
We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in vi... more We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in visual transduction in Limulus photoreceptors. Although phosphoinositide hydrolysis is central to phototransduction and phosphoinositide-dependent Ca(2+)-mobilization seems to be required for transduction, the subsequent steps leading to ion channel gating (the immediate cause of excitation) are not understood. Channels normally opened in response to light can be opened in excised membrane patches by cGMP but not by Ca2+, suggesting that cGMP acts as a channel ligand in excitation. Using phosphodiesterase inhibitors, we investigated whether changes in cGMP metabolism could affect excitation. We report that zaprinast and IBMX increased the amplitudes and retarded the kinetics of physiological light responses. These effects were maximal for brightest stimuli. The effects were markedly enhanced in low Ca2+ conditions. In contrast, excitation induced by direct IP3-injection and by direct Ca(2+...
Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition ... more Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the group III receptors mGluR4, mGluR6, and mGluR7. [3H]L-AP4 binding was present in human embryonic kidney cells transfected with the high-affinity mGluR4 receptor but not in cells transfected with mGluR6 or the low-affinity mGluR7 receptor. Analysis of mGluR4/mGluR6 receptor chimeras revealed that replacement of the first 35 amino acids of mGluR6 with the first 50 amino acids of mGluR4 was sufficient to impart [3H]L-AP4 binding to mGluR6. Homology models of mGluR4 and mGluR7 were used to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high-affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, whereas the N74K mutation in combination with mutations in residues 258 to 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared with unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the group III mGluRs.
The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain... more The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naı̈ve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.
Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of... more Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of nicotinic agonists improve attention, and nicotinic antagonist exposure impairs it. This study was conducted to investigate the effect of a novel nicotinic receptor agonist at α4β2 nicotinic receptors (AZD3480) on attention and reversal of pharmacologically induced attentional impairment produced by the NMDA glutamate antagonist dizocilpine (MK-801). Adult female Sprague-Dawley rats were trained to perform an operant visual signal detection task to a stable baseline of accuracy. The rats were then injected subcutaneously following a repeated measures, counter-balanced design with saline, AZD3480 (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations 30 min before the test. The effect of donepezil on the same pharmacologically induced attentional impairment was also tested. A separate group of rats was injected with donepezil (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations, and their attention were assessed. Saline was the vehicle control. Dizocilpine caused a significant (p < 0.0005) impairment in percent correct performance. This attentional impairment was significantly (p < 0.0005) reversed by 0.01 and 0.1 mg/kg of AZD3480. AZD3480 by itself did not alter the already high baseline control performance. Donepezil (0.01-1.0 mg/kg) also significantly (p < 0.005) attenuated the dizocilpine-induced attentional impairment. AZD3480, similar to donepezil, showed significant efficacy for counteracting the attentional impairment caused by the NMDA glutamate antagonist dizocilpine. Low doses of AZD3480 may provide therapeutic benefit for reversing attentional impairment in patients suffering from cognitive impairment due to glutamatergic dysregulation and likely other attentional disorders.
We have cloned two splice variants of the human homolog of the ␣ 1A subunit of voltage-gated Ca 2... more We have cloned two splice variants of the human homolog of the ␣ 1A subunit of voltage-gated Ca 2ϩ channels. The sequences of human ␣ 1A-1 and ␣ 1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human ␣ 1A-2 ␣ 2b ␦ 1b Ca 2ϩ channels expressed in HEK293 cells activate rapidly ( ϩ10mV ϭ 2.2 ms), deactivate rapidly ( -90mV ϭ 148 s), inactivate slowly ( ϩ10mV ϭ 690 ms), and have peak currents at a potential of ϩ10 mV with 15 mM Ba 2ϩ as charge carrier. In HEK293 cells transient expression of Ca 2ϩ channels containing ␣ 1A/B(f) , an ␣ 1A subunit containing a 112 amino acid segment of ␣ 1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba 2ϩ currents that were 30-fold larger compared to wild-type (wt) ␣ 1A-2containing Ca 2ϩ channels, and had inactivation kinetics similar to those of ␣ 1B-1 -containing Ca 2ϩ channels. Cells transiently transfected with ␣ 1A/B(f) ␣ 2b ␦ 1b expressed higher levels of the ␣ 1 , ␣ 2b ␦, and  1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N 1655 P 1656 ) and S5-SS1 (E 1740 ) that influence the biophysical properties of ␣ 1A . ␣ 1A E1740R resulted in a threefold increase in current magnitude, a Ϫ10 mV shift in steady-state inactivation, and an altered Ba 2ϩ current inactivation, but did not affect ion selectivity. The deletion mutant ␣ 1A ⌬NP shifted steady-state inactivation by Ϫ20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by -Aga IVA was increased with ␣ 1A ⌬NP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of ␣ 1A -containing Ca 2ϩ channels.
Electrophysiological investigations were carried out on several independently isolated mutants of... more Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1- 6 photoreceptors, and a mutant of theninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodop- sin content in R1-6 photoreceptors is reduced by 10 2-106-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologi- cally normal . Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model . Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps . Mutant bumpsare strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletio...
The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by... more The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by the second messenger, cyclic GMP. Injection of cGMP into rods or application of cGMP to inside-out membrane patches activates a conductance resembling that produced by light. Light produces a rapid reduction of cGMP in living rods, leading to closure of sodium channels and membrane hyperpolarization. In most invertebrate photoreceptors the response to light is depolarizing. We have investigated whether cGMP is involved in controlling the increase in sodium conductance that underlies this depolarization. We show here that injection of cGMP into Limulus photoreceptors produces a depolarization that mimics the receptor potential. We also show that the cGMP concentration of the squid retina increases rapidly during exposure to light. These results support the hypothesis that cGMP mediates the light-induced depolarization in invertebrate photoreceptors and suggests that vertebrate and invertebrate phototransduction may be more similar than previously thought.
In this review we have discussed the problem of deactivation at both the rhodopsin and G protein ... more In this review we have discussed the problem of deactivation at both the rhodopsin and G protein levels. Of particular interest is the novel observation that rhodopsin deactivation can be modulated by light. This modulation is likely to play an important role in light adaptation by reducing the gain of transduction. One interesting possibility is that this modulation involves the phosphorylation of an arrestin-like molecule, but this remains to be tested. One of the experimental advantages of Limulus photoreceptors is the large size of the single photon responses and the fact that even single G proteins produce a detectable response. This made possible the observation that nonhydrolyzable GTP analogues produce discrete transient events rather than the step-like events that would be predicted by previous models. This observation led us to a new view of how enzyme deactivation is coupled to GTP hydrolysis on G protein. According to this view, enzymes are activated by G protein, but ca...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1995
We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in vi... more We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in visual transduction in Limulus photoreceptors. Although phosphoinositide hydrolysis is central to phototransduction and phosphoinositide-dependent Ca(2+)-mobilization seems to be required for transduction, the subsequent steps leading to ion channel gating (the immediate cause of excitation) are not understood. Channels normally opened in response to light can be opened in excised membrane patches by cGMP but not by Ca2+, suggesting that cGMP acts as a channel ligand in excitation. Using phosphodiesterase inhibitors, we investigated whether changes in cGMP metabolism could affect excitation. We report that zaprinast and IBMX increased the amplitudes and retarded the kinetics of physiological light responses. These effects were maximal for brightest stimuli. The effects were markedly enhanced in low Ca2+ conditions. In contrast, excitation induced by direct IP3-injection and by direct Ca(2+...
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Papers by Edwin Johnson