Isolation and heterologous expression of two genomic clones encoding Shaker-related potassium channels of trout CNS

Two Shaker-related potassium channel genes (termed tsha1 and tsha2) express ed in the CNS of trout were cloned and sequenced. The coding regions of bot h genes were not interrupted by introns and exhibited a high overall sequen ce similarity to other members of the Shaker subfamily. By computer-assiste d sequence alignments, tsha1 was identified as a fish homologue to the mamm alian Kv1.2 subtype of potassium channels, whereas tsha2 did not show a pre ferential sequence homology but shared a uniform similarity to Kv1.1, Kv1.2 , and Kv1.3. Upon heterologous expression in a mammalian glial cell line, b oth channels exhibited delayed rectifier current properties that differed f rom each other by their threshold potentials of activation and their pharam cological features: wheras the tsha1-mediated current was efficiently block ed by submicromolar concentrations of alpha-DTX but not by TEA, tsha2 was h ighly TEA-sensitive, correlating well with differences in the amino acid st ructure of the pore outer mouth region. As revealed by RT-PCR, Shaker-relat ed potassium channels were sequentially expressed during trout brain develo pment: tsha2 was found initially at stage 34, followed by tsha1 at stage 36 , whereas two other members of the Shaker family (tsha3 and tsha4) were det ectable much earlier (stage 30, hatching). In the mature brain tissue, no r egional specialization of Shaker channel subtype expression was noted. (C) 2000 Wiley-Liss, Inc.