Figure 3

α ENaC alternatively spliced forms. A schematic illustration of alternative mRNA splicing of α ENaC wildtype, -a and -b forms. α ENaC wildtype is made of 12 exons, while α ENaC-a is formed of exons I to VIII, with a 23 bases deleted from exon VIII. On the other hand, α ENaC-b is formed of exons I to IX with a skipping of exon VIII [79 bases]. Underneath each mRNA splicing comes the protein organization of the 2 alternatively spliced forms of α ENaC [α ENaC-a & -b] that have been published in rats [33] in comparison to α ENaC wildtype major transcript. α ENaC wildtype is 698 amino acids in length [2100 bp]. Amino acid residues from 1 to 110 reside in the cytoplasm, amino acid residues from 111 to 131 constitutes the first transmembrane domain, residues 132 to 589 constitute the extracellular loop, residues 590 to 610 constitute the second transmembrane domain, and residues 611-698 are cytoplasmic. α ENaC-a alternatively spliced form is formed by the deletion of 23 nucleotides from exon 8, whereas α ENaC-b is formed by the deletion of 79 nucleotides that involved exon 8 skipping. These deletions introduced a premature stop codon and resulted in shorter proteins at the carboxy terminus by 199 in α ENaC-a and 216 amino acids in α ENaC-b, making α ENaC-a 499 amino acids [2077 bp] and α ENaC-b 482 amino acids [2021 bp] in length. These resultant shorter proteins lacked the second transmembrane domain [TDM2] which is important in channel pore formation. α ENaC-a alternatively spliced form is a low abundance transcript that is expressed in the rat kidney, tongue epithelia and tongue taste tissues. α ENaC-a binding with the channel blocker [phenamil] was greatly enhanced. This demonstrates that the amiloride-binding site [i.e ENaC blocker site] resides in the extracellular loop of the channel and not the second transmembrane domain that is presently missing in α ENaC-a [CD: cytoplasmic domain, TDM1: transmembrane domain M1, EC: extracellular loop, TDM2: transmembrane domain M2].