Expression of Kir7.1 and a novel Kir7.1 splice variant in native human retinal pigment epithelium.
Previous studies on bovine retinal pigment epithelium (RPE) established that Kir7.1 channels compose this epithelium's large apical membrane K+ conductance. The purpose of this study was to determine whether Kir7.1 and potential Kir7.1 splice variants are expressed in native adult human RPE and, if so, to determine their function and how they are generated. RT-PCR analysis indicated that human RPE expresses full-length Kir7.1 and a novel Kir7.1 splice variant, designated Kir7.1S. Analysis of the human Kir7.1 gene (KCNJ13) organization revealed that it contains three exons, two introns, and a novel alternative 5' splice site in exon 2. In human RPE, the alternative usage of two competing 5' splice sites in exon 2 gives rise to transcripts encoding full-length Kir7.1 and Kir7.1S, which is predicted to encode a truncated protein. Real-time PCR indicated that Kir7.1 transcript is nearly as abundant as GAPDH mRNA in human RPE whereas Kir7.1S transcript expression is 4-fold lower. Western blot analysis showed that the splice variant is translated in Xenopus oocytes injected with Kir7.1S cRNA and revealed the expression of full-length Kir7.1 but not Kir7.1S in adult human RPE. Co-expression of Kir7.1 with Kir7.1S in Xenopus oocytes had no effect on either the kinetics or amplitude of Kir7.1 currents. This study confirms the expression of Kir7.1 in human RPE, identifies a Kir7.1 splice variant resulting in predicted changes in protein sequence, and indicates that there is no functional interaction between this splice variant and full-length Kir7.1.
Department of Ophthalmology and Visual Sciences, University of Michigan, W.K. Kellogg Eye Center, 1000 Wall St., University of Michigan, Ann Arbor, MI 48105, USA.
Dongli
D
Yang
Anuradha
A
Swaminathan
Xiaoming
X
Zhang
Bret A
BA
Hughes
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008252:Macaca fascicularis; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014981:Xenopus
D005136:Eye Proteins; C489477:Kir7.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger