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Abstract In the present study, the effects of rutaecarpine on ionic currents of NG108-15 neuronal cells were studied. Rutaecarpine (2-100 £gM) suppressed the amplitude of voltage-dependent K+ outward current (IK(DR)) in a concentration-dependent manner. The IC50 value for rutaecarpine-induced inhibition of IK was 11£gM. However, rutaecarpine (20 £gM) had little effect on L-type Ca2+ current. IK(DR) present in these cells is sensitive to the inhibition by quinidine and dendrotoxin, yet not by E-4031. Rutaecarpine enhanced the rate and extent of IK(DR) inactivation, although it had no effect on the initial activation phase of IK(DR). Recovery from block by rutaecarpine (5 £gM) was fitted by a single exponential with a value of 2.87 s. Cell-attached single-channel recordings revealed that rutaecarpine decreased channel activity over the length of the test potential without altering single-channel amplitude. With the aid of binding scheme, a quantitative description of the actions of rutaecarpine on IK(DR) was provided. Under current-clamp configuration, rutaecarpine also prolonged action potential duration in NG108-15 cells without altering other variables of the action potential. The results clearly show that rutaecarpine is a blocker of the KDR channel. The increase in action potential duration induced by rutaecarpine can be explained mainly by its blocking effects on IK(DR).

Successful synaptic transmission at the neuromuscular junction depends on the precise alignment of the nerve terminals with the postsynaptic specialization of the muscle fiber. It is increasingly apparent that this precision is achieved during development and maintained in the adult through signals exchanged between motoneurons and their target muscle fibers that serve to coordinate their spatial and temporal differentiation. There is increasing evidence that retinoic acid (RA), a derivative of Vitamin A, plays important roles in the development of nervous system. Here we specifically test this notion by examining the effect of RA on synaptic activity at developing neuromuscular synapse in Xenopus cell culture using whole-cell patch clamp recording. Bath application of RA rapidly (with a 10~15 min latency) and specifically enhances the spontaneous ACh secretion and the action of ¡@RA is reversible. The synaptic potentiation induced by RA was not occluded in the presence of protein synthesis inhibitor, Anisomycin and Cycloheximide,¡@suggesting this is transcription-independent. Selective RAR

Activation of a5??1 integrin potentiates L-type calcium current in vascular smooth muscle, which is partly mediated by tyrosine phoshorylation of the a1c channel subunit. Expressed rabbit VSM and neuronal isoforms are also potentiated by a5??1 integrin activation and require dual phosphorylation of a1c by PKA and c-Src. To explore common mechanisms of regulation by a5??1 integrin, whole cell patch clamp experiments were used to investigate the effects of a5??1 integrin antibody on expressed cardiac calcium channels. In HEK cells transfected with a1c, ??2a and a2-d1 subunits alone, currents increased 1.8 ?? 2.0 fold on application of a5??1 antibody. The potentiation was almost completely abolished on the application of PKI, a highly specific Protein Kinase A (PKA) inhibitor. The expressed currents increased 2.0 ?? 2.2 fold on application of PKA activator 8-Br-cAMP, and abolished by PKI. Our results suggest that regulation of L-type calcium channels by a5??1 integrin is a general mechanism shared by VSM, neuronal and cardiac channels. However, in the cardiac isoform, only PKA phosphorylation is involved.

1 / Yanyan Jiang

The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.

Diss.--Chimie--Göteborg--Université de Göteborg, 2001. / Bibliogr. p. 63-70. Index.

This thesis presents a neuron emulator and headstage circuit for patch clamp setups and provides simulation, measurement and verification results. The circuit implemented on a printed circuit board (PCB) is battery powered and portable. The emulator provides both passive (resting potential) and active (action potential) electrical properties of a live neuron as seen from a single electrode by using the headstage circuit. It can be used to test electrophysiological equipment such as current-clamp, voltage-clamp or patch-clamp amplifiers. The action potentials (APs) are generated with a voltage-dependent frequency controlled by a microcontroller implementing a firing range from -60 mV to -30 mV and firing frequency from 1 Hz to10 Hz. The charge released by firing the neuron is initially stored on a 110 pC capacitor. Compared to directly using a current or voltage source, this design results in a more realistic simulation of the APs generated by ionic currents in a live neuron. The measured results from a prototype demonstrate that the neuron emulator meets the design specifications and it is capable of performing voltage clamp and rate responsive current clamp functionality. Measured results using a commercial clamp amplifier are provided to confirm the emulator operation in a practical recording environment.

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No description available.