Cell-specific patterns of alternative splicing of voltage-gated ion channels in single identified neurons

Abstract

CbNav and CbIH encode channels that carry voltage-gated sodium and hyperpolarization activated cation currents respectively in the crab, Cancer borealis. We cloned and sequenced full length cDNAs for both CbNav and CbIH and found nine different regions of alternative splicing for the CbNav gene and four regions of alternative splicing for CbIH. We used RT-PCR to determine tissue-specific differences in splicing of both channel genes among cardiac muscle, skeletal muscle, brain, and stomatogastric ganglion (STG) tissue. We then examined the splice variant isoforms present in single, unambiguously identified neurons of the STG. We found cell-type specific patterns of alternative splicing for CbNav, indicating unique cell-specific pattern of post-transcriptional modification. Furthermore, we detected possible differences in cellular localization of alternatively spliced CbNav transcripts; distinct mRNA isoforms are present between the cell somata and the axons of the neurons. In contrast, we found no qualitative differences among different cell types for CbIH variants present, although this analysis did not represent the full spectrum of all possible CbIH variants. CbIH mRNA was not detected in axon samples. Finally, although cell-type specific patterns of splicing were detected for CbNav, the same cell type within and between animals also displayed variability in which splice forms were detected. These results indicate that channel splicing is differentially regulated at the level of single neurons of the same neural network, providing yet another mechanism by which cell-specific neuronal output can be achieved.