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Volume 17, Number 5, Issue of March 1, 1997 pp. 1582-1595
Copyright ©1997 Society for NeuroscienceIdentification of a Vesicular Pool of Calcium Channels in the Bag Cell Neurons of Aplysia californica
Received Oct. 4, 1996; revised Dec. 11, 1996; accepted Dec. 16, 1996.
Benjamin H. White and Leonard K. Kaczmarek Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
To study the molecular mechanism of calcium current modulation in the bag cell neurons of Aplysia californica, we have identified calcium channel subtypes expressed in these cells and analyzed their distribution using channel-specific antibodies. Using PCR to amplify reverse-transcribed RNA from bag cell clusters, we identified two classes of calcium channel
1 subunit. One, BCCa-I, belongs to the ABE subfamily of calcium channels, whereas the other, BCCa-II, belongs to the SCD subfamily. Antibodies generated against the bag cell calcium channels recognize membrane proteins of ~210 and 280 kDa on immunoblots. Both channels are expressed in the bag cell clusters as well as in other parts of the Aplysia nervous system. BCCa-II also localizes to glia and muscle. The subcellular distribution of the two channel types is strikingly different. Antibody staining of bag cell neurons in primary culture shows that BCCa-II is present on the plasma membrane, whereas BCCa-I has a punctate, intracellular distribution consistent with a vesicular localization. The BCCa-I-containing vesicles are found in bag cell neuron somata and growth cones and occasionally in neuritic hotspots. Their distribution is similar but not identical to that of LysoTracker Red, a marker for acidic organelles, but unlike that of dense-core vesicles containing egg-laying hormone. The vesicular channels may represent the protein kinase C-sensitive calcium channels of bag cell neurons that are believed to enhance hormonal release during electrical activity.
Key words: calcium channel; bag cell neuron; Aplysia; protein kinase C; vesicle; neuroendocrine; invertebrate; immunocytochemistry; antibody; glia
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