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D J Baro

Publications and source records attributed to D J Baro.

12 recordsLinked to original sources
PubMed2000

Molecular underpinnings of motor pattern generation: differential targeting of shal and shaker in the pyloric motor system.

The patterned activity generated by the pyloric circuit in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, results not only from the synaptic connectivity between the 14 component neurons but also from differences in the intrinsic properties of the neurons. Presumably, differences in the complement and distribution of expressed ion channels endow these neurons with many of their distinct attributes. Each pyloric cell type possesses a unique, modulatable transient potassium current, or A-current (I(A)), that is instrumental in determining the output of the network. Two genes encode A-channels in this system, shaker and shal. We examined the hypothesis that cell-specific differences in shaker and shal channel distribution contribute to diversity among pyloric neurons. We found a stereotypic distribution of channels in the cells, such that each channel type could contribute to different aspects of the firing properties of a cell. Shal is predominantly found in the somatodendritic compartment in which it influences oscillatory behavior and spike frequency. Shaker channels are exclusively localized to the membranes of the distal axonal compartments and most likely affect distal spike propagation. Neither channel is detectably inserted into the preaxonal or proximal portions of the axonal membrane. Both channel types are targeted to synaptic contacts at the neuromuscular junction. We conclude that the differential targeting of shaker and shal to different compartments is conserved among all the pyloric neurons and that the channels most likely subserve different functions in the neuron.

Amino Acid Sequence↗
PubMed2000

Patterns of shaker family gene expression in single identified neurons of the American lobster, Homarus americanus.

The patterns of expression of voltage gated potassium channel genes of the Shaker family have been mapped in identified neurons of the lobster (Homarus americanus) ventral nerve cord using a single cell reverse transcriptase polymerase chain reaction procedure. Using specific oligonucleotides derived from the sequences of the shaker, shab, and shaw genes of the spiny lobster, Panulirus interruptus, we detected the corresponding potassium channel DNA fragments from Homarus americanus. The Homarus DNA fragments are 87-98% identical at the nucleotide level to the Panulirus DNA fragments. We used the Panulirus primers to measure the complement of RNAs for shaker, shab, and shaw in single identified cells that use GABA, glutamate, octopamine or serotonin as chemical messengers. Shaker and shaw RNAs were found in all four identified neuron types but shab RNA was not detected in serotonin cells under the present experimental conditions. All cells expressed alpha-tubulin RNA, which serves as an internal control suggesting that cells are intact after dissection. In glial cells that surround the neuronal cell bodies, the potassium channel genes are expressed at low to non-detectable levels.

Animals↗
PubMed1998

Differential expression and targeting of K+ channel genes in the lobster pyloric central pattern generator.

A molecular analysis of motor pattern generation is an essential complement to electrophysiological and computational investigations. In arthropods, A-channels are posttranslationally modified multimeric proteins containing Shaker family alpha-subunits that may interact with beta-subunits, gamma-subunits, and other auxiliary proteins. One consequence of A-channel structure is that several mechanisms could underlie the cell-specific differences in pyloric IAs including differential gene expression, alternate splicing, and posttranslational modifications. Oocyte expression studies, single-cell RT-PCR, and immunocytochemistry suggest that differential alpha-subunit gene expression is not a mechanism for creating pyloric IA heterogeneity, and that the same gene, shal, encodes the alpha-subunits for the entire family of somatic IAs in the pyloric network. Changes in the level of shal gene expression alter A-channel density between cells, but cannot account for the differences in the biophysical properties of the six pyloric IAs. Preliminary data suggest that the shal gene also encodes the A-channel alpha-subunits for the coarse and fine neuropil but not for most axons. A second gene, shaker, encodes the A-channel alpha-subunits in the majority of axons and at the neuromuscular junction. The distinct properties of the two types of A-channels are consistent with the different roles of IA at the different locations. Both the shaker and shal genes are alternately spliced, and investigations are under way to determine whether alternate splicing is a mechanism for generating pyloric IA heterogeneity.

Animals↗
PubMed1998

Expression of Panulirus shaker potassium channel splice variants.

In Drosophila shaker voltage-dependent potassium channels, alternative splicing at the amino and carboxy termini produces currents with different electrophysiological characteristics. We have cloned alternatively spliced forms of shaker from the spiny lobster Panulirus interruptus. Alternative exons were found at three sites of the gene; eight different 5' exons, two alternative exons encoding the pore-forming P region, and an alternative 3' exon. Two of the different amino terminal splice forms were expressed with two alternatively spliced pore forms to produce channels with markedly different characteristics. One of the amino termini produced a channel with transient characteristics while the other produced a delayed rectifier-type channel. The effects of alternative exons at the amino terminus and in the P region appear to be additive. Our results provide new information on the structural requirements for rapid N-type inactivation.

Alternative Splicing↗
PubMed1997

Alternative splicing in the pore-forming region of shaker potassium channels.

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 5' and 3' 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, kappa-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+ currents or synaptic transmission between neurons.

Amino Acid Sequence↗
PubMed1997

Quantitative single-cell-reverse transcription-PCR demonstrates that A-current magnitude varies as a linear function of shal gene expression in identified stomatogastric neurons.

Different Shaker family alpha-subunit genes generate distinct voltage-dependent K+ currents when expressed in heterologous expression systems. Thus it generally is believed that diverse neuronal K+ current phenotypes arise, in part, from differences in Shaker family gene expression among neurons. It is difficult to evaluate the extent to which differential Shaker family gene expression contributes to endogenous K+ current diversity, because the specific Shaker family gene or genes responsible for a given K+ current are still unknown for nearly all adult neurons. In this paper we explore the role of differential Shaker family gene expression in creating transient K+ current (IA) diversity in the 14-neuron pyloric network of the spiny lobster, Panulirus interruptus. We used two-electrode voltage clamp to characterize the somatic IA in each of the six different cell types of the pyloric network. The size, voltage-dependent properties, and kinetic properties of the somatic IA vary significantly among pyloric neurons such that the somatic IA is unique in each pyloric cell type. Comparing these currents with the IAs obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster Ishaker and lobster Ishal, respectively) reveals that the pyloric cell IAs more closely resemble lobster Ishal than lobster Ishaker. Using a novel, quantitative single-cell-reverse transcription-PCR method to count the number of shal transcripts in individual identified pyloric neurons, we found that the size of the somatic IA varies linearly with the number of endogenous shal transcripts. These data suggest that the shal gene contributes substantially to the peak somatic IA in all neurons of the pyloric network.

Animals↗
PubMed1996

The lobster shaw gene: cloning, sequence analysis and comparison to fly shaw.

We cloned and sequenced the cDNA for the shaw gene, encoding a voltage-dependent potassium (K+) channel, from the spiny lobster, Panulirus interruptus. The deduced amino acid sequence has a high degree of homology to the Drosophila melanogaster Shaw protein. In addition, lobster Shaw has several putative sites for post-translational modifications.

Amino Acid Sequence↗
PubMed1996

Lobster shal: comparison with Drosophila shal and native potassium currents in identified neurons.

The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyte expression system.

Amino Acid Sequence↗
PubMed1996

RT-PCR analysis of shaker, shab, shaw, and shal gene expression in single neurons and glial cells.

We have developed a reverse transcription-polymerase chain reaction (RT-PCR) method to examine single neurons and glial cells in the stomatogastric ganglion of the spiny lobster Panulirus interruptus for the expression of four members of the Shaker family of potassium channel genes. Using this technique we found that shaker, shab, shaw, and shal are expressed in 100%, 78%, 100%, and 94% of stomatogastric neurons. Furthermore, neuronal shab, shaw, and shal transcript levels vary among cells in a manner which is independent of cell size. We also detected Shaker family gene expression in glial cells. Shaker, shaw, and shal are detectably expressed in 100%, 63%, and 100% of the glial caps examined, respectively, while shab gene expression could not be detected in glial cells.

Animals↗
PubMed1994

Shab gene expression in identified neurons of the pyloric network in the lobster stomatogastric ganglion.

A single shab gene exists in the lobster, Panulirus interruptus, and undergoes alternate splicing to produce multiple transcripts. Using in situ hybridization we have determined the expression pattern of the shab gene in identified neurons of the pyloric network. The shab gene is consistently expressed at a low level in the Ventricular Dilator cell, a high level in the Pyloric Dilator cell, and is not detectably expressed in the Lateral Pyloric or Inferior Cardiac cells. Shab gene expression in the Anterior Burster cell varies from animal to animal. The electrophysiologically heterogeneous group of eight Pyloric Constrictor cells also shows differences in shab gene expression. These results support the idea that differences in shab gene expression contribute to the unique electrophysiological phenotypes displayed by each cell type.

Amino Acid Sequence↗
PubMedDate not supplied

An improved parameter estimation method for Hodgkin-Huxley models.

We consider whole-cell voltage-clamp data of isolated currents characterized by the Hodgkin-Huxley paradigm. We examine the errors associated with the typical parameter estimation method for these data and show them to be unsatisfactorally large especially if the time constants of activation and inactivation are not sufficiently separated. The size of these errors is due to the fact that the steady-state and kinetic properties of the current are estimated disjointly. We present an improved parameter estimation method that utilizes all of the information in the voltage-clamp conductance data to estimate steady-state and kinetic properties simultaneously and illustrate its success compared to the standard method using simulated data and data from P. interruptus shal channels expressed in oocytes.

Animals↗