Yang D et al. (2008), Expression of Kir7.1 and a novel Kir7.1 splice ...

XB-ART-36877

Exp Eye Res 2008 Jan 01;861:81-91. doi: 10.1016/j.exer.2007.09.011.

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Expression of Kir7.1 and a novel Kir7.1 splice variant in native human retinal pigment epithelium.

Yang D , Swaminathan A , Zhang X , Hughes BA .

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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.

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Species referenced: Xenopus
Genes referenced: kcnj13

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	Fig. 1. Expression of Kir7.1 and Kir7.1S in human RPE and other tissues. Expression of Kir7.1 and Kir7.1S in native human (A,C,D) and monkey (B) RPE, neural retina (Ret), human kidney (Ki), and brain (Br). RT reactions were performed in the presence (+) or absence (−) of reverse transcriptase. PCR was performed using a primer set specific for both Kir7.1 and Kir7.1S (see Table 1). M, DNA size markers.
	Fig. 2. Kir7.1S nucleotide and protein sequences. (A) Sequences of Kir7.1 and Kir7.1S cDNA derived from native human RPE. Start and stop codons are indicated by bold underlined letters. Deleted region in the Kir7.1S sequence is indicated by short bars. (B) Alignment of amino acid sequences of Kir7.1 and Kir7.1S protein from native human RPE. Lowercase letters indicate differences created by a shift in reading frame for Kir7.1S caused by 236-bp deletion of nucleotides corresponding to 244–479 of Kir7.1 cDNA. M1 and M2 represent two transmembrane segments and P the pore-forming P-region.
	Fig. 3. PCR of human genomic DNA. PCR was performed with primer set 1 (A: lanes 1 and 2), primer set 2 (A: lanes 3 and 4), primer set 3 (B: lanes 1–3), or primer set 4 (B: lanes 4–6) using 100 ng human genomic DNA (A: lanes 1 and 3; B: lanes 1, 2, 4 and 5) or water (A: lanes 2 and 4; B: lanes 3 and 6) as template. M1, M2, and M, DNA size markers. The sizes of the products amplified by primer sets 1, 2, and 3 match those predicted for KCNJ13. The failure of Kir7.1S-specific primer set 4 to amplify a product indicates that a distinct gene encoding Kir7.1S does not exist.
	Fig. 4. The exon-intron organization of human Kir7.1 gene (KCNJ13). (A) The exon-intron organization of human Kir7.1 gene determined by comparing a published human genomic DNA sequence (GenBank accession no. AC064852), the Kir7.1 (GenBank accession no. AY758241) and Kir7.1S (GenBank accession no. AY758240) cDNA sequences revealed in this study, and a published Kir7.1 cDNA sequence (GenBank accession no. NM_002242) using Lasergene software (DNASTAR) and information obtained from AceView (http://www.humangenes.org). Diagonal lines indicate introns and boxes represent exons. The novel alternative donor splice site is located within the second exon. (B) Alignment of consensus sequences of splice sites with Kir7.1 and Kir7.1S sequences. Bold lowercase letters indicate GT and AG base pairs for donor and acceptor sites. M represents A or C; r represents a or g.
	Fig. 5. Quantitation of Kir7.1 and Kir7.1S mRNA levels in native human RPE. (A) Amplification plots of Kir7.1 (green), Kir7.1S (red) and GAPDH (dark blue) in native human RPE. The y-axis represents the fluorescence intensity and the x-axis the PCR cycle number. (B) Melting curves of Kir7.1 (green), Kir7.1S (red) and GAPDH (dark blue). (C) Agarose gel electrophoresis of the expression of Kir7.1, Kir7.1S and GAPDH in native human RPE. (D) Summary of the relative expression of Kir7.1 and Kir7.1S relative to GAPDH in the RPE obtained from the five donors. *P < 0.01 compared to Kir7.1.
	Fig. 6. Expression of human Kir7.1S protein in Kir7.1S cRNA-injected Xenopus oocytes and native human RPE. Western blot of Xenopus oocyte protein and human RPE lysate probed with anti-Kir7.1 antibody alone (left panel) or with antibody preabsorbed with antigenic peptide (right panel). Lane 1, uninjected Xenopus oocytes; lane 2, Xenopus oocytes injected with Kir7.1S; and lane 3, native human RPE. Anti-Kir7.1 N-terminal antibody recognized Kir7.1S protein in Xenopus oocytes (lane 2) but only Kir7.1 in human RPE (lane 3).
	Fig. 7. Functional characterization of Kir7.1 expressed alone and together with Kir7.1S in Xenopus oocytes. Current–voltage (I–V) relationships of whole-cell currents recorded in the presence of extracellular ND96 (2 mM K+) and 98 mM K+ or 98 mM Rb+ solution in a Kir7.1 cRNA-injected oocyte (A and B) and an oocyte injected with equal amounts of Kir7.1 and Kir7.1S cRNAs (C and D). (E) Summary of current amplitudes in Kir7.1- and Kir7.1:Kir7.1S-injected oocytes measured at −125 mV in the presence of ND96, 98 mM K+, and 98 mM Rb+ (mean ± SD, n = 5 for each group).

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