These results indicate that the Kir7.1 channel subunit, but not Kir4.1, is a major component of the apical K(+) conductance in bovine RPE. Kir7.1 is distributed over the length of apical processes, where it probably functions in the regulation of K(+) transport and the electrical response of the RPE to light-evoked changes in subretinal K(+) concentration.

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.

To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2. 4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1-q13.3. Expression of human Kir2. 4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K(+) currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K(+) channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.

Inwardly rectifying K+ (Kir) channels in the apical membrane of the retinal pigment epithelium (RPE) contribute to extracellular K+ homeostasis in the distal retina by mediating K+ secretion. Multiple lines of evidence suggest that these channels are composed of Kir7.1. Previously, we showed that native Kir channels in bovine RPE are modulated by changes in intracellular pH in the physiological range. In the present study, we used the Xenopus laevis oocyte expression system to investigate the pH dependence of cloned human Kir7.1 channels and several point mutants involving histidine residues in the NH2 and COOH termini. Kir7.1 channels were inhibited by strong extracellular acidification and modulated by intracellular pH in a biphasic manner, with maximal activity at about intracellular pH (pHi) 7.0 and inhibition by acidification or alkalinization. Replacement of histidine 26 (H26) in the NH2 terminus with alanine eliminated the requirement of protons for channel activity and increased sensitivity to proton-induced inhibition, resulting in maximal channel activity at alkaline pHi and smaller whole cell currents at resting pHi compared with wild-type Kir7.1. When H26 was replaced with arginine, the pHi sensitivity profile was similar to that of the H26A mutant but with the p Ka shifted to a more acidic value, giving rise to whole cell current amplitude at resting pHi that was comparable to that of wild-type Kir7.1. These results indicate that Kir7.1 channels are modulated by intracellular protons by diverse mechanisms and suggest that H26 is important for channel activation at physiological pHi and that it influences an unidentified proton-induced inhibitory mechanism.

Although there are many advanced technologies and techniques for silicon diagnostics, effective failure analysis to root cause is getting increasingly challenging, as very often the electrical failure analysis data would point to a symptom that is the result of the defect rather than the actual location of the defect. Therefore, a combination of multiple techniques is often employed so that sensitivity of "the cause of the problem" can be observed. This work compiles a successful analysis with the aid of continuous wave laser voltage probing and soft defect localization techniques and presents three cases that are voltage-sensitive fails. The first case is a 28 nm device which failed at-speed scan. The second case is a 28 nm device failing RAM register BIST with high Vmin and the third case is a scan shift failure in a less than 28nm device.