Interaktion des ROMK-Kaliumkanals mit dem ABCB1 (ATP-Binding Cassete Typ B1) Protein

Zusammenfassung In der Entstehung von hereditären Bartter-ähnlichen Salzverlusttubulopathien spielt der einwärtsgleichrichtende ROMK-Kalium-Kanal mit Sitz im dicken aufsteigenden Teil der Henle-Schleife eine entscheidende Rolle. Insbesondere die enge funktionale Kopplung der verschiedenen Transport...

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Bibliographic Details
Main Author: Hilpert, Johannes
Contributors: Waldegger, Siegfried (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2010
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ABSTRACT Physiological interaction of ROMK with the ABCB1-protein (ABC-transporter B1) Hereditary salt-losing tubular disorders are rare but life-threatening genetic deseases. Early diagnosis and therapeutic intervention prevent patients fatal outcome. Prenatal diagnosis may reveal up to 60% of underlying genetic defects. While searching for new candidate genes, we identified ABCB1 in Yeast-Two-Hybrid studies as a ROMK interacting protein. The Renal Outer Medullary Potassium Channel (ROMK) is crucial for the process of salt absorption in the thick ascending limb of Henle’s loop (TAL). A functional defective ROMK channel results in severe, prenatal onset renal salt wasting accompagnied by maternal polyhydramnios, prematurity, and hypovolemia showing the phenotype of the antenatal Bartter-Syndrome. Therefore ROMK-interacting partners may be promising candidates, which could be responsible for genetically unexplained salt wasting tubular disorders. In this study the physiological interaction of ROMK and ABCB1 is characterised in Xenopus laevis oocytes as a heterologous expression system by using the Two-Electrode-Voltage-Clamp method (TEVC-method) and a luminometric assay for surface quantification studies. Results revealed a strong effect of ABCB1 on ROMK mediated whole-cell currents when coexpressed in Xenopus Oocytes. ABCB1 causes a massive inhibition (up to 100%) of the ROMK-dependent whole cell current. The inhibition depends upon injected ABCB1 m-RNA concentrations. ABCB1 coexpression did not inhibit currents mediated by Kir2.1, an inward rectifier potassium channel closely related to ROMK. These findings indicate the interaction to be specific for ROMK and ABCB1. Additionally surface-quantification-studies with a heamagglutinin tagged ROMK-Protein (ROMK-HA) were carried out. The studies showed a 60% reduction of surface signal, when ROMK-HA was coinjected with ABCB1 compared to ROMK-injection alone. We conclude that ABCB1 interferes with ROMK trafficking towards the cell membrane. However, an additional effect of ABCB1 on ROMK channel gating can not be excluded. Other ABC-transporter proteins have been shown to interact with ATP-dependent K+ inward-rectifier (Kir) channels. One well known example is the interaction of Kir6.2 with the sulfonyl urea receptor (SUR) in the pancreatic β-cell. Both, channel and ABCmolecule, form an heterooctamer which is the molecular basis of the ATP-sensitive β-cell potassium conductance. Coexsistence of at least two distinct apical K+ channels in the TAL, a 35 pS (lowconductance)channel and a 70 pS (intermediate-conductance) channel, has been shown. Functional characterisation of isolated expressed ROMK was compatible with the native 35 pS K+ channel. Whereas isolating the 70 pS K+ channel failed in the past. In addition to that ROMK-deficient mice neither express the 35 pS K+-channel nor the 70 pS K+-channel. These findings strongly suggest the 70 pS K+-channel to be composed out of ROMK and an accessory protein which may serve as a β-subunit. Our studies indicate a possible interaction between ROMK and ABCB. If the partners do or do notheteromerise to build a functional channel e.g. similar to the Kir6.2 and SUR interaction, remains unclear. Despite the attenuating effect of ABCB1 on ROMK generated whole cell currents it still might be possible that some functional heteromeric channels reach the plasma membrane. Additionaly certain limitatons should be considered when using Xenopus Oocytes, such as low incubation temperature or possible interaction between endogenously and heterologously expressed proteins. Regarding this, a possible influence of ABCB1 on renal salt-and water reabsorption requires further investigation. This includes studies in human cell lines or e.g. knockout mice studies. Although these observations were made independently and not in direct comparison, it should be noted that there is a phenotypical concordance between ROMK and ABCB knock out mice concerning several features of renal concentration capabilities and salt wasting. Both types of knockout mice show increased diuresis, lower renal plasma flow combined with lower glomerular filtration rate, as well as an augmented urinary excretion of sodium and calcium along with poor concentration capabilities. If ABCB1 should be proofen to function as a distinct modulating element in renal saltand water reabsorption, genomic DNA samples of patients could be screened to define genetically unexplained salt wasting tubular disorders. Beyond that our findings may possibly contribute to the understanding of the overall process of renal salt absorption.