Deliverables
The molecular mechanisms underlying the functional properties of acetylcholine receptors (AChR) were analyzed by 5 laboratories (from St. Petersburg and Puschino, Russia; Kiev, Ukraine; Rome, Italy; and Paris, France) using a combination of biophysics, molecular biology and chemistry methods.
The project was developed in three main directions:
Analysis of AChR modulation by second messengers. Functional properties of AChR-operated channels expressed in Xenopus oocytes and human cell lines (HEK 293 and BOSC).Evaluation relations between receptor operated channels and neuronal cytoskeleton.
AChR modulation by second messengers were studied in non-dissociated neurons of rat superior cervical ganglion (SCG) and guinea-pig submucous plexus (SMP) using a whole-cell patch clamp and two-electrode voltage clamp recording methods. Thapsigargin, the inhibitor of microsomal calcium-ATPase, strongly suppressed the ACh currents in SCG and SMP neurons, not affecting the EPSC amplitude and the EPSC decay time constant. Forscolin, the adenylate cyclase activator, did not affect AChR in both ganglia. Genistein, the tyrosine protein kinase inhibitor, did not affect the ACh current in SCG neurons, but strongly suppressed it in SMP neurons. The results suggest that the activity of the postsynaptic nAChRs in the SCG neurons is strongly suppressed by raised intracellular Ca2+ level, and by increased protein kinase C activity, through the mechanisms not involving tyrosine kinase, protein kinase A and protein kinase G.
Mono-and two-ammonium compounds with long aliphatic chains were synthesized for comparison of their blocking effects on neuronal nicotinic AChRs. Two of them (IEM1678 and IEM-1556) selectively blocked nicotinic cholinergic transmission in the intracardiac parasympathetic ganglia in vivo. The results of the study suggest that these compounds can be recommended for the treatment of heart rate disturbances caused by vagus nerve hyperactivity.
For evaluation functional properties of nAChRs, cDNAs coding for different subunits were expressed in Xenopus oocytes and human cell lines (HEK 293 or BOSC) and studied using electrophysiology and fluorescence techniques. It was shown that α6 subunit forms functional Ca2+ permeable nAChRs in combination with ß4 and may coassemble with α3 and ß4 nAChR subunits to form a triplet receptor. Functional receptors are formed also by the coexpression of α6 and ß2 subunits. Moreover α5 subunit when coexpressed in transfected cells with α3 and ß4 participates in the formation of α3ß4α5 nAChRs. The fractional calcium currents of tested nAChRs varies between 2 % and 5 % of the total current. A potent stimulator of protein kinase C (TPA) potentiated ACh-induced currents and caused acceleration of its decay. It suggests that modulation of AChR currents may be developed independently from phosphorylation of either γ or δ subunit.
Relations between receptor operated channels and neuronal cytoskeleton were studied in dialyzed neurons from snail Lymnaea stagnalis using voltage-clamp method. The data suggests that depolymerization of microfilaments cause acceleration of AChR currents decay.
Finally, using confocal scanning microscopy and patch-clamp methods direct monitoring of cytoskeletal microfilaments in hippocampal and cerebellar granule neurons was conducted. Activation of N-methyl-D-aspartate receptor (NMDA)-activated ionic channels accompanied by Ca2+ influx and by a decrease in measured fluorescein-phalloidin fluorescence. These data provide a direct evidence that elevation of cytoplasmic Ca2+ can cause depolymerization of F-actin in neurons.
Results of this study are described in 8 papers and several abstracts.
The molecular mechanisms underlying the functional properties of acetylcholine receptors (AChR) were analyzed by 5 laboratories (from St. Petersburg and Puschino, Russia; Kiev, Ukraine; Rome, Italy; and Paris, France) using a combination of biophysics, molecular biology and chemistry methods.
The project was developed in three main directions:
Analysis of AChR modulation by second messengers. Functional properties of AChR-operated channels expressed in Xenopus oocytes and human cell lines (HEK 293 and BOSC).Evaluation relations between receptor operated channels and neuronal cytoskeleton.
AChR modulation by second messengers were studied in non-dissociated neurons of rat superior cervical ganglion (SCG) and guinea-pig submucous plexus (SMP) using a whole-cell patch clamp and two-electrode voltage clamp recording methods. Thapsigargin, the inhibitor of microsomal calcium-ATPase, strongly suppressed the ACh currents in SCG and SMP neurons, not affecting the EPSC amplitude and the EPSC decay time constant. Forscolin, the adenylate cyclase activator, did not affect AChR in both ganglia. Genistein, the tyrosine protein kinase inhibitor, did not affect the ACh current in SCG neurons, but strongly suppressed it in SMP neurons. The results suggest that the activity of the postsynaptic nAChRs in the SCG neurons is strongly suppressed by raised intracellular Ca2+ level, and by increased protein kinase C activity, through the mechanisms not involving tyrosine kinase, protein kinase A and protein kinase G.
Mono-and two-ammonium compounds with long aliphatic chains were synthesized for comparison of their blocking effects on neuronal nicotinic AChRs. Two of them (IEM1678 and IEM-1556) selectively blocked nicotinic cholinergic transmission in the intracardiac parasympathetic ganglia in vivo. The results of the study suggest that these compounds can be recommended for the treatment of heart rate disturbances caused by vagus nerve hyperactivity.
For evaluation functional properties of nAChRs, cDNAs coding for different subunits were expressed in Xenopus oocytes and human cell lines (HEK 293 or BOSC) and studied using electrophysiology and fluorescence techniques. It was shown that α6 subunit forms functional Ca2+ permeable nAChRs in combination with ß4 and may coassemble with α3 and ß4 nAChR subunits to form a triplet receptor. Functional receptors are formed also by the coexpression of α6 and ß2 subunits. Moreover α5 subunit when coexpressed in transfected cells with α3 and ß4 participates in the formation of α3ß4α5 nAChRs. The fractional calcium currents of tested nAChRs varies between 2 % and 5 % of the total current. A potent stimulator of protein kinase C (TPA) potentiated ACh-induced currents and caused acceleration of its decay. It suggests that modulation of AChR currents may be developed independently from phosphorylation of either γ or δ subunit.
Relations between receptor operated channels and neuronal cytoskeleton were studied in dialyzed neurons from snail Lymnaea stagnalis using voltage-clamp method. The data suggests that depolymerization of microfilaments cause acceleration of AChR currents decay.
Finally, using confocal scanning microscopy and patch-clamp methods direct monitoring of cytoskeletal microfilaments in hippocampal and cerebellar granule neurons was conducted. Activation of N-methyl-D-aspartate receptor (NMDA)-activated ionic channels accompanied by Ca2+ influx and by a decrease in measured fluorescein-phalloidin fluorescence. These data provide a direct evidence that elevation of cytoplasmic Ca2+ can cause depolymerization of F-actin in neurons.
Results of this study are described in 8 papers and several abstracts.
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