| Abstrakt: |
To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel α subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel α subunits, including the S4 region involved in activation, the pore loops responsible for ion selectivity, and the inactivation loop between the third and fourth domains, though the latter lacks the highly conserved IFM motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based real time RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 355371, 2003 [ABSTRACT FROM AUTHOR] |
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