ABSTRACT
The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP(3) pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting.
Subject(s)
Abnormalities, Multiple/genetics , Ear/abnormalities , Mice, Mutant Strains/genetics , Mutation, Missense/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Skull/abnormalities , Amino Acid Sequence , Animals , Base Sequence , Calcium/metabolism , Immunohistochemistry , Mice , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
1 P2Y receptors are expressed in the nervous system and are involved in calcium signalling in neurons and glia. In the superior cervical ganglion (SCG), RT-PCR analysis indicated the presence of P2Y(1,2&6) receptors. Rises in intracellular calcium in response to P2Y receptor stimulation were determined from adult mouse cultured SCG neurons and glia. 2 ADP evoked suramin (100 microM)- and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM)-sensitive rises in intracellular calcium in approximately 80% of SCG neurons (EC50 approximately 20 microM). ADP-evoked responses were abolished in neurons from P2Y1 receptor-deficient mice (responses to UTP were unaffected). 3 The pyrimidines UTP (EC50 approximately 85 microM) and UDP (EC50>90 microM) evoked PPADS- and suramin-sensitive responses in approximately 70 and approximately 20% of SCG neurons, respectively. 4 In SCG glial cells, ADP (EC50 approximately 30 microM) evoked calcium responses in approximately 50% of glia. These were suramin and PPADS sensitive and essentially abolished in SCG glial cells cultured from adult P2Y1 receptor-deficient mice. 5 UTP (EC50 approximately 25 microM) and UDP (EC50>200 microM) evoked suramin- and pyridoxalphosphate-6-azophenyl-2',5'-disulphonate-sensitive rises in calcium in approximately 60 and 20% SCG glial cells, respectively. 6 These results indicate the presence of several P2Y receptors coupled to an increase in intracellular calcium in the SCG: ADP-sensitive P2Y1 receptors and UDP-sensitive P2Y6 receptors in SCG neurons and glial cells, a novel UTP-sensitive P2Y receptor in SCG neurons and UTP- and ATP-sensitive P2Y2 receptors in SCG glia.
Subject(s)
Calcium/metabolism , Neuroglia/metabolism , Neurons/metabolism , Pyridoxal Phosphate/analogs & derivatives , Receptors, Purinergic P2/physiology , Superior Cervical Ganglion/metabolism , Uridine Triphosphate/pharmacology , Animals , Cells, Cultured , Female , Fluorescent Dyes , Male , Mice , Mice, Inbred C57BL , Neuroglia/drug effects , Neurons/drug effects , Pyridoxal Phosphate/pharmacology , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y1 , Receptors, Purinergic P2Y2 , Reverse Transcriptase Polymerase Chain Reaction , Superior Cervical Ganglion/cytology , Superior Cervical Ganglion/drug effects , Suramin/pharmacology , Uridine Diphosphate/pharmacologyABSTRACT
The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole-cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPgammaS (100 microm) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 microm). alpha,beta-meATP also facilitated sEPSC and sIPSC frequency, while l-beta,gamma-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPgammaS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 microm) and the selective P2X(3) receptor antagonist A-317491 (3 microm), suggesting that modulation of sEPSCs involves P2X(3) receptor subunits. alpha,beta-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X(1) receptor-deficient mice demonstrating a functional role for P2X(1) receptors in the CNS.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Brain Stem/physiology , Excitatory Postsynaptic Potentials/physiology , Neural Inhibition/physiology , Receptors, Purinergic P2/physiology , Synapses/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Brain Stem/drug effects , Cell Line , Excitatory Postsynaptic Potentials/drug effects , Humans , In Vitro Techniques , Mice , Mice, Knockout , Neural Inhibition/drug effects , Protein Subunits/agonists , Protein Subunits/antagonists & inhibitors , Protein Subunits/deficiency , Protein Subunits/physiology , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Rats , Receptors, Purinergic P2/deficiency , Synapses/drug effectsABSTRACT
P2X receptors are highly expressed throughout the nervous system, where ATP has been shown to be a neurotransmitter. The aim of this study was to characterize P2X receptor expression within sympathetic postganglionic neurons from the superior cervical ganglia. Reverse transcription-polymerase chain reaction showed the presence of mRNA for all P2X receptors, raising the possibility of multiple subunit expression within these ganglia. Whole-cell patch-clamp and calcium imaging studies revealed a heterogeneous population of P2X receptors in approximately 70% of neurons. We propose that the heterogeneity in properties could be caused by differential expression and/or subunit composition of the P2X receptor. The dominant phenotype was P2X2-like; neurons showed slow desensitization, sensitivity to antagonists, and a profile of ionic modulation that is characteristic of P2X2 receptors: potentiation by acidification and extracellular Zn2+ and attenuation by high extracellular Ca2+ and pH. A subpopulation of neurons (10-15%) were alpha,beta-methylene ATP (alpha,beta-meATP) sensitive, and in neurons from P2X1 receptor-deficient mice the alpha,beta-meATP response was reduced to 2% of all neurons, demonstrating a direct role for P2X1 subunits. Control alpha,beta-meATP responses were eliminated by high extracellular Ca(2+) and pH, indicating the presence of heteromeric channels incorporating the properties of P2X1 and P2X2 receptors. This study demonstrates that in neurons, the P2X1 receptor can contribute to the properties of heteromeric P2X receptors. Given the expression of P2X1 receptors in a range of neurons, it seems likely that regulation of the properties of P2X receptors by this subunit is more widespread.
