Structural requirements for rapid inactivation and voltage dependence in splice variants of lobster Shaker potassium channels.

We studied the properties of currents generated in Xenopus oocytes by nine splice variants of the spiny lobster Shaker gene. These isoforms differ in their amino termini and in the P-loop region of the pore. Both the voltage dependence and kinetic properties of the currents varied significantly, depending on which amino terminus was present. A cluster of net positive charges at the N-terminus was not necessary for rapid inactivation: negatively charged N-termini also inactivated rapidly. There was no obvious correlation between N-terminus length and inactivation rate. These N-terminal effects were additive with a separate set of voltage and kinetic properties controlled by the two alternative P-loop exons.

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.

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.