Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents

Pfaffinger, Paul J.; Furukawa, Yasuo; Zhao, Biao; Dugan, Deborah; Kandel, Eric R.

doi:10.1523/jneurosci.11-04-00918.1991

Cited by 68 publications

(57 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been proposed that the AKv1.1a gene product forms the ion channels for native I Adepol measured in Aplysia neurons (Pfaffinger et al, 1991). Furukawa (1995) described pronounced statedependent, cumulative inactivation of the AKv1.1a channels expressed in frog oocytes.…”

Section: Cumulative Inactivation Of I Adepol Potentiated By a Positmentioning

confidence: 99%

“…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).…”

Section: Cumulative Inactivation Of I Adepol Potentiated By a Positmentioning

confidence: 99%

See 1 more Smart Citation

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

Koester²

1996

J. Neurosci.

View full text Add to dashboard Cite

The R20 neurons of Aplysia exhibit frequency-dependent spike broadening. Previously, we had used two-electrode voltage clamp to examine the mechanisms of this spike broadening (Ma and Koester, 1995). We identified three K ϩ currents that mediate action-potential repolarization: a transient A-type K ϩ current (I Adepol ), a delayed rectifier current (I K-V ), and a Ca 2ϩ -sensitive K ϩ current (I K-Ca ). A major constraint in that study was the lack of completely selective blockers for I Adepol and I K-V , resulting in an inability to assess directly the effects of their activation and inactivation on spike broadening. In the present study, the dynamic-clamp technique, which employs computer simulation to inject biologically realistic currents into a cell under current-clamp conditions (Sharp et al., 1993a,b), was used either to block I Adepol or I K-V or to modify their inactivation properties.The data in this paper, together with earlier results, lead to the following hypothesis for the mechanism of spike broadening in the R20 cells. As the spike train progresses, the primary responsibility for spike repolarization gradually shifts from I Adepol to I K-V to I K-Ca . This sequence can be explained on the basis of the relative rates of activation and inactivation of each current with respect to the constantly changing spike durations, the cumulative inactivation of I Adepol and I K-V , and the progressive potentiation of I K-Ca . Positive feedback interactions between spike broadening and inactivation contribute to the cumulative inactivation of both I Adepol and I K-V . The data also illustrate that when two or more currents have similar driving forces and partially overlapping activation characteristics, selectively blocking one current under current-clamp conditions can lead to a significant underestimate of its normal physiological importance.Key words: spike broadening; dynamic clamp; K ϩ current; I Adepol ; inactivation; Aplysia ; R20 Frequency-dependent spike broadening, an endogenously generated increase in spike duration that increases as a function of firing rate, has been shown in neurons from a variety of species to be correlated with enhanced transmitter release (Gillette et al., 1980;Coates and Bulloch, 1985) and to result primarily from inactivation of K ϩ currents (Aldrich et al., 1979a,b;Jackson et al., 1991;Bielefeldt et al., 1992;Crest and Gola, 1993;Quattrocki et al., 1994). Previously we had described the facilitatory synaptic effects and the mechanisms of frequency-dependent spike broadening in the two electrically coupled peptidergic R20 neurons of Aplysia (Ma and Koester, 1995). In addition, to analyze the mechanisms underlying spike broadening, several voltageactivated currents were isolated from the R20 cells by conventional voltage-clamp methods. These included a Na ϩ current (I Na ), a multi-component Ca 2ϩ current (I Ca ), and three K ϩ currents-a high-threshold transient A-type current (I Adepol ), a delayed rectifier current (I K-V ), and a two component Ca 2ϩ -sensitive K ϩ current...

show abstract

Section: Cumulative Inactivation Of I Adepol Potentiated By a Positmentioning

confidence: 99%

Section: Cumulative Inactivation Of I Adepol Potentiated By a Positmentioning

confidence: 99%

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

Koester²

1996

J. Neurosci.

View full text Add to dashboard Cite

show abstract

“…I A is not a uniform current, but it can be expressed with different properties (Wei et al, 1990). Different neurons can show markedly different types of I A , with varying maximal conductance, voltage dependence, and degree and rate of inactivation (Pfaffinger et al, 1991;Furakawa et al, 1992;Baro et al, 1996b). This helps neurons to have different intrinsic electrophysiological properties.…”

mentioning

confidence: 99%

Alternative Splicing in the Pore-Forming Region ofshakerPotassium Channels

et al. 1997

View full text Add to dashboard Cite

We have cloned cDNAs for the shaker potassium channel gene from the spiny lobster Panulirus interruptus. As previously found in Drosophila, there is alternative splicing at the 5Ј and 3Ј ends of the coding region. However, in Panulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, -conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, and Panulirus shal were not blocked. When it was tested in the Panulirus stomatogastric ganglion, the toxin produced no effects on transient K ϩ currents or synaptic transmission between neurons.

show abstract

“…With this expression vector, the reporter gene lacZ from Escherichia coli is expressed in 80%o of injected cells. Using this vector, we have been able to analyze the properties and physiological roles of a cloned Aplysia Shaker K+ channel (AKOla) (14). We find that AKOla phenocopies the native K+ current IAdepoi, suggesting that this native K+ channel is assembled as a homooligomer of AKOla.…”

mentioning

confidence: 99%

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

Kaang

Pfaffinger

Grant

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents

Cited by 68 publications

References 30 publications

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

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

Alternative Splicing in the Pore-Forming Region ofshakerPotassium Channels

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

Contact Info

Product

Resources

About

Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents

Cited by 68 publications

References 30 publications

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

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

Alternative Splicing in the Pore-Forming Region ofshakerPotassium Channels

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

Contact Info

Product

Resources

About

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

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