First, we determined which pre-exposure BK mRNA variants are expressed in the naïve SON, using end-point polymerase chain reaction (PCR). BK has at least six alternative splice sites residing within the coding sequence (Beisel et al., 2007, Figure 1A). Using primers unique to each splice site in rat brain, we found alternatively spliced exons at only three (X3, X4, and X6) of these sites (Figure 1, and Figure S1 in the Supplemental Materials). Next, using primers bracketing sites X3 through X6, we observed several distinct bands (Figure 1B), suggesting the expression of several distinct variants of BK, possibly with different exonal concatenations. Indeed, subcloning and sequencing revealed that these bands corresponded to splice variants with differing exonal concatenations (Figure 1C). We observed only eight of the possible sixteen variants, indicating that exonal assembly of BK message within SON neurons is not random. Exon 29 located at site X6 was most frequent.
