Overexpression of an Aplysia shaker K+ channel gene modifies the electrical properties and synaptic efficacy of identified Aplysia neurons.

Abstract

Although potassium channels play a variety of roles in shaping the electrical properties of neurons, little is known about how these channels are constituted in neurons. To examine the assembly and physiological function of A-type K+ channels in mature differentiated neurons, we have developed a higly efficient gene transfer method forAplysia neurons that has allowed us to express about 107 copies of the cloned Aplysia Shaker (Sh) K+ channel (AKOla) in single identified cells. We find that expression of AKOla phenocopies one of the native transient K+ currents (IAepa)J suggesIng that the native channel carrying IAd&pI is assembled as a homooligomer of AKOla. Overexpression of AKOla has substantial effect on the action potential, shortening its duration, enhancing its hyperpolarizing afterpotential, and depressing by more than half the amount of transmitter release by the action potential from the terminals. Thus, the AKOla channel not only contributes to the firing properties within a given neuron but also can regulate the signaling between interconnected cells.Potassium channels are important regulators of the resting potential, the action potential, and the excitability of neurons (1). In addition, they have a role in certain forms of procedural learning. For example, in the marine molluscs Aplysia (2-5) and Hermissenda (6-9) modulation of different species of K+ currents in sensory neurons contributes to several learned modifications of simple reflex behaviors. The cloning of the Shaker (Sh) locus in Drosophila (10-13) has now made it possible to study the functions of various K+ channels on the molecular level. However, K+ channels have so far not been successfully transfected into mature, differentiated nerve cells in the intact nervous system. As a result, it has not been possible to study cloned K+ channels in their native cellular environment and to determine, by detailed comparison of cloned and endogenous channels, the subunit composition of any endogenous K+ channel. G. R. MacGregor, Houston, TX) as a BamHI fragment (3.7 kb) and inserted into the BamHI site of pNEX, and the orientation was determined. To make pNEX-AKO1a, the full-length cDNA fragment (1.8 kb) of Aplysia Shaker (AKOla) having Cla I and Xmn I sites at 5' and 3' ends, respectively, was inserted into Acc I-and Sma I-digested pNEX.Microinjection of Plasmids into Aplysia Neurons. From Aplysia californica weighing 10-100 g (furnished by the Howard Hughes Medical Institute, Miami Facility) the abdominal ganglion was dissected and treated with protease IX (Sigma) at 10 mg/ml in L15 medium (15) for 1 hr at 34.50C.The ganglion was then pinned on a Sylgard plate and desheathed carefully to expose the neurons to the L15 medium containing an equal volume of Aplysia hemolymph or artificial seawater (ASW) that had the following composition: 460 mM NaCl/10 mM KCl/11 mM CaCl2/55 mM MgCl2/10 mM Hepes, pH 7.6. In a few experiments, neurons in primary cell culture were used. Primary cell culture techniques and media have been described (15). Withi...

The Role of K+Currents in Frequency-Dependent Spike Broadening inAplysiaR20 Neurons: A Dynamic-Clamp Analysis

“…This difference in inactivation properties may indicate that the AKv1.1a gene product is not a component of the I Adepol channels. More likely, given the other strong similarities between native I Adepol and I AKv1.1a (Pfaffinger et al, 1991;Kaang et al, 1992), they differ in their inactivation properties either because of differences in ion composition or post-translational modification between oocytes and Aplysia neurons or because the AKv1.1a channels lack unidentified ␤-subunits or heterologous ␣-subunits that may be present in native I Adepol channels (Sheng et al, 1993;Rettig et al, 1994).…”

“…A drawback to this method is the possible difficulty in obtaining accurate descriptions of several voltage-sensitive conductances. A third approach is to express artificially the channel in a neuron in which it does not normally appear (Kaang et al, 1992) and then to observe the resultant changes in excitability properties. This approach is quite demanding technically.…”

Analysis of genes encoding highly conserved lysine‐rich proteins in Aplysia californica and Saccharomyces cerevisiae

“…The isolation of its promoter from a genomic library will be necessary to confirm this possibility. This could represent a good alternative to the R14 or viral promoters already described in Aplysia (DesGroseillers et al, 1987;Kaang et al, 1992). However, we do not exclude the possibility that the gene may be regulated in Aplysia, as described below in yeast.…”

“…Several properties of identified Aplysia neurons, such as the size of their cell body and the extensive mapping of the neuronal pathways (Kandel, 1979) make them an ideal system for such a purpose, Due to the large size of the neurons, it is possible to microinject DNA or mRNA to change the phenotype of the cells and/or to test the function of molecules acting on neuronal circuits (DesGroseillers et al, 1987;Mochida et al, 1990;Dash et al, 1990;Kaang et a]., 1992). We demonstrated that endogenous neuropeptide promoter sequences driving neuropeptide precursor genes or the chloramphenicol acetyl transferase reporter gene can be successfully microinjected into Aplysia giant neurons.…”

Nitric oxide modulation of calcium‐activated potassium channels in postganglionic neurones of avian cultured ciliary ganglia

“…Over-expression of potassium channels has been shown to lead to an increase in the rate of repolarization of the action potential in nerve terminals, with a consequent decrease in the duration of the action potential and in calcium influx, so decreasing transmitter release (Kaang et al, 1992). Repolarization of the action potential is in part due to the opening of fast calcium-activated potassium channels (Ij) in sympathetic neurones (Lancaster & Pennefather, 1987;Marsh & Brown, 1991) and in the hip-pocampus (Lancaster & Adams, 1986;Storm, 1987;Yoshida et al, 1991).…”