Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay

Abstract

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gα_s-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gα_s and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl⁺ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl⁺ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.

Keywords: High-throughput screening; Intracellular signaling; Inward rectifying K+ channels (Kir7.1); Melanocortin 4 receptor (MC4R); Thallium flux assay.

Fig. 1

Sample plate map: calculation of the ΔF_Ratio necessitates the use of a checkerboard plate map. F_Ratio traces from agonist-treated well traces are subtracted from the vehicle-treated trace directly above (odd columns) or below (even columns) to obtain ΔF_Ratio. We have found that 12–16 compound well/vehicle subtractions, or two columns, are necessary to detect significant differences in Kir7.1 flux

Fig. 2

Experimental protocol and sample traces: (a) A cell plate is prepared with 2 × 10⁴ cells/well in a 384-well plate and incubated for 24 h. Cells are then washed with assay buffer, loaded with dye buffer, and incubated for 30 min. After another wash with assay buffer, the ligand is added using a 384-well robotic pipettor to ensure that every well receives agonist at the same time. After 20 min, the plate is now ready to be read in the FDSS kinetic plate reader. (b) Once placed in the FDSS reader, a 2 min baseline reading (F₀) is obtained. Using the FDSS automated liquid handler, 10 μL of a 5 × Thallium buffer is added to each well of the assay plate. Fluorescence is read for 10 min following this addition. ΔF_Ratio is then calculated using the method described in Subheading 3.3

Fig. 3

α-MSH reduces Kir7.1-mediated thallium flux: (a) HEK 293 T cells expressing MC4R and Kir7.1 plated in a 384-well plate are exposed to a 20 min incubation of 100 nM α-MSH in a checkerboard pattern. This agonist causes a reduction in ΔF_Ratio following the addition of thallium. Gray average of ΔF_Ratio measurements, Error bars ± SEM n = 110. (b) Using the ΔF_Ratio a Z-factor of 0.19 was throughput assay. n = 110

Fig. 4

AgRP increases Kir7.1 mediated thallium flux: 100 nM AgRP increases thallium flux in HEK293 cells expressing MC4R and Kir7.1. Gray average of ΔF_Ratio measurements, Error bars ± SEM. n = 36

Fig. 5

Plate map strategy for analyzing compounds that modulate MC4R-Kir7.1 signaling: This plate map format allows the study of pharmocological probes in the thallium flux assay. In this example, Rp-cAMPs, a potent competitive inhibitor of the activation of cAMP-dependent protein kinases, is dispensed in a checkerboard format on columns 5 and 6 but in every well of columns 7 and 8. Following a 10 μL addition of this compound to the assay plate, a second 10 μL stimulus of 400 nM (4 ×) α-MSH is then added. This plate is designed in a checkerboard format on columns 3 and 4 for a positive control and columns 7 and 8 to determine the ability of Rp-cAMPs to block the effect of α-MSH

Fig. 6

MC4R-Kir7.1 signaling is not regulated by Gs: (a) 100 μM Rp-cAMPs does not block the effect of α-MSH on Kir7.1 conductance. n = 36 ΔF_Ratio measurements/group; Error bars ± SEM; One Way ANOVA p < 0.001. (b) In this experiment, a dominant negative Gs plasmid or a wild-type Gs plasmid is transfected into HEK293 cells expressing MC4R and Kir7.1. Forty-eight hours later, cells are exposed to a dose response curve of α-MSH. Expression of a dominant negative Gs plasmid in MC4R-Kir7.1 cells does not right shift the α-MSH dose response curve as indicated by the lack of an effect on the EC₅₀ of α-MSH. n = 12 ΔF_Ratio measurements/group; error bars ± SEM; One Way ANOVA p < 0.001