Detection of transcripts for delayed rectifier potassium channels in the Xenopus laevis inner ear

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify sequences for delayed rectifier potassium (drk) channel transcripts in Xenopus laevis inner ear and brain. We used degenerate primers that spanned a region between the N-terminal cytoplasmic portion and a region located between the S2 and S3 transmembrane domains of the potassium channel protein. When inner ear total RNA or brain mRNA was used as a template for RT-PCR, a unique product of the expected size (approximately 560 bp) was observed as a single band after electrophoresis on agarose gels. The PCR product from reactions using X. laevis genomic DNA as template was similarly sized, indicating a lack of introns in this region. The RT-PCR products from inner ear and brain were isolated, cloned, and sequenced. Sequence analysis showed that the X. laevis inner ear and brain clones were identical. Sequence alignments of the cloned RT-PCR products with posted GenBank sequences established that the drk sequences from X. laevis inner ear and brain share highest identity with larval X. laevis brain, mouse, rat, and human Kv2 sequences. Positive signals were obtained from inner ear and brain mRNA in Northern dot blots hybridized with digoxigenin labeled probes from the inner ear clone. Taken together, results provide evidence for the expression of Kv2 sequences in the X. laevis inner ear and brain.