The localization of two voltage-gated calcium channels in the pyloric network of the lobster stomatogastric ganglion.

Voltage-gated calcium channels are critical to all aspects of nervous system function, with differing roles within the neuronal somata, at synaptic terminals, and at the neuromuscular junction. We have developed antibodies against two voltage-gated Ca(2+) channel genes from the spiny lobster, Panulirus interruptus, which are homologous to the Drosophila Ca1A (a P/Q-type channel) and Ca1D (an L-type channel) genes. Using these antibodies, we have found that each channel shows unique patterns of localization within the stomatogastric nervous system. Both antibodies stain somata of most of the neurons in the pyloric network to varying degrees. The high degree of variability in staining intensity within individual pyloric cell classes supports the hypothesis of Golowasch et al. (1999a,b) that individual cells can vary in their composition of ionic currents and still have similar firing properties. Anti-Ca1A stains structures in the neuropil, some of which are terminals of axons descending from higher ganglia; however, the majority of these are neither neurites nor blood vessels, but may instead be glial cells or other support elements. Anti-Ca1A labeling was also prominent in the peripheral axons of pyloric motoneurons as they enter muscles, indicating that this channel may be involved in regulation of synaptic transmission onto the foregut muscles. Anti-Ca1D does not label neurites in the neuropil of the stomatogastric ganglion. It stains glial cells in the stomatogastric ganglion in the region of their nuclei, presumably from protein being produced in the perinuclear rough endoplasmic reticulum, en route to the glial cell periphery. While anti-Ca1D labeling is seen in a patchy distribution along peripheral pyloric axons, it was never seen near the muscle. We conclude that the localization of these two calcium channels is tightly controlled within the stomatogastric nervous system, but we cannot conclusively demonstrate that Ca1A and/or Ca1D channels play roles in synaptic integration within the stomatogastric ganglion.

Cellular localization of Shab and Shaw potassium channels in the lobster stomatogastric ganglion.

The motor pattern generated by the 14 neurons composing the pyloric circuit in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, is organized not only by the synaptic connections between neurons, but also by the characteristic intrinsic electrophysiological properties of the individual cells. These cellular properties result from the unique complement of ion channels that each cell expresses, and the distribution of those channels in the cell membranes. We have mapped the STG expression of shab and shaw, two genes in the Shaker superfamily of potassium channel genes that encode voltage-dependent, non-inactivating channels. Using antibodies developed against peptide sequences from the two channel proteins, we explored the localization and cell-specific expression of the channels. Anti-Shab and anti-Shaw antibodies both stain all the pyloric neurons in the somata, as well as their primary neurites and branch points of large neurites, but to varying degrees between cell types. Staining was weak and irregular (Shaw) or absent (Shab) in the fine neuropil of pyloric neurons, where most synaptic interactions occur. There is a high degree of variability in the staining intensity among neurons of a single cell class. This supports Golowasch et al.'s [J Neurosci 19 (1999) RC33; Neural Comput 11 (1999) 1079] hypothesis that individual cells can have similar firing properties with varying compositions of ionic currents. Both antibodies stain the axons of the peripheral nerves as they enter foregut muscles. We conclude that both Shab and Shaw channels are appropriately localized to contribute to the noninactivating potassium current in the stomatogastric nervous system.

Alternate splicing of the shal gene and the origin of I(A) diversity among neurons in a dynamic motor network.

The pyloric motor system, in the crustacean stomatogastric ganglion, produces a continuously adaptive behavior. Each cell type in the neural circuit possesses a distinct yet dynamic electrical phenotype that is essential for normal network function. We previously demonstrated that the transient potassium current (I(A)) in the different component neurons is unique and modulatable, despite the fact that the shal gene encodes the alpha-subunits that mediate I(A) in every cell. We now examine the hypothesis that alternate splicing of shal is responsible for pyloric I(A) diversity. We found that alternate splicing generates at least 14 isoforms. Nine of the isoforms were expressed in Xenopus oocytes and each produced a transient potassium current with highly variable properties. While the voltage dependence and inactivation kinetics of I(A) vary significantly between pyloric cell types, there are few significant differences between different shal isoforms expressed in oocytes. Pyloric I(A) diversity cannot be reproduced in oocytes by any combination of shal splice variants. While the function of alternate splicing of shal is not yet understood, our studies show that it does not by itself explain the biophysical diversity of I(A) seen in pyloric neurons.

KChIP1 and frequenin modify shal-evoked potassium currents in pyloric neurons in the lobster stomatogastric ganglion.

The transient potassium current (I(A)) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I(A) in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I(A) had somewhat different properties from the endogenous I(A). The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopus oocytes or lobster PD neurons, they produced A-currents resembling the endogenous I(A) in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca(2+) sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.

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.

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.

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.

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.