Measuring Electroneutral Chloride-Dependent Ion Fluxes in Mammalian Cells and in Heterologous Expression Systems

Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

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Kv3.1–Kv3.2 Channels Underlie a High-Voltage–Activating Component of the Delayed Rectifier K+ Current in Projecting Neurons From the Globus Pallidus

Journal of Neurophysiology ◽

10.1152/jn.1999.82.3.1512 ◽

1999 ◽

Vol 82 (3) ◽

pp. 1512-1528 ◽

Cited By ~ 64

Author(s):

R. Hernández-Pineda ◽

A. Chow ◽

Y. Amarillo ◽

H. Moreno ◽

M. Saganich ◽

...

Keyword(s):

Heterologous Expression ◽

Globus Pallidus ◽

High Frequency ◽

Calcium Binding ◽

Repetitive Firing ◽

Delayed Rectifier ◽

Expression Systems ◽

Electrophysiological Properties ◽

Channel Proteins ◽

Heterologous Expression Systems

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3.1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than −10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1–Kv3.2 voltage-gated K+channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.

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