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A novel fluorescence‐based assay of ROMK1 K + channel function
Author(s) -
Denton Jerod,
Weaver David
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb42-a
Subject(s) - hek 293 cells , chemistry , potassium channel , transfection , patch clamp , biophysics , sk channel , microbiology and biotechnology , heterologous expression , biochemistry , recombinant dna , ion channel , biology , receptor , gene
ROMK (Kir1.1) plays key physiological roles in maintenance of fluid and electrolyte homeostasis by the kidney. Pharmacological modulation of ROMK activity could be useful for the management of hypertension and edema. However, the value of ROMK as a therapeutic target is unexplored due in part to the lack of selective ROMK channel modulators. The identification of novel ROMK modulators is therefore highly desirable, but currently available assays of ROMK function are not amenable to high‐throughput screening of chemical compound libraries. Therefore, our objective is to develop a fluorescence‐based assay of ROMK channel activity for the purpose of high‐throughput discovery of novel ROMK modulators. As a first step toward this goal, HEK293 cells were transiently transfected with ROMK1 carrying a serine‐to‐aspartate mutation of residue 44 (R‐S44D) to promote cell surface expression of the channel. Patch clamp analysis showed that heterologous expression of R‐S44D in HEK293 cells produces robust K + ‐selective currents that are blocked by Ba 2+ and tertiapin‐Q (TPNQ). To test if R‐S44D activity can be measured with optical methods, transfected cells were loaded with the thallium (Tl + )‐sensitive fluorescent dye BTC. TPNQ‐sensitive Tl + flux through R‐S44D was detected in transfected cells plated in 384‐well plates. These pilot studies indicate that ROMK channel activity can be measured using the Tl + ‐flux assay. We are currently optimizing the assay and generating stable cells lines to maximize the fluorescence signal‐to‐noise ratio. With further development, we are optimistic that this assay will permit high‐throughput discovery of novel ROMK channel modulators.