−/− ventricular myocytes. We provide evidence that this change in action potential shape leads to an increased driving force for the L-type Ca 2+ current during the action potential, which explains the altered contractility of the heart muscle. Conclusions:

TRPM4 is a Ca(2+)-activated nonselective cation channel. The channel is activated by an increase of intracellular Ca(2+) and is regulated by several factors including temperature and Pi(4,5)P2. TRPM4 allows Na(+) entry into the cell upon activation, but is completely impermeable to Ca(2+). Unlike TRPM5, its closest relative in the transient receptor potential family, TRPM4 proteins are widely expressed in the body. Currents with properties that are reminiscent of TRPM4 have been described in a variety of tissues since the advent of the patch clamp technology, but their physiological role is only beginning to be clarified with the increasing characterization of knockout mouse models for TRPM4. Furthermore, mutations in the TRPM4 gene have been associated with cardiac conduction disorders in human patients. This review aims to overview the currently available data on the functional properties of TRPM4 and the current understanding of its physiological role in healthy and diseased tissue.

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a Ki value of 111±16 nM in radioligand binding using [3H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A2AAR antagonist. MRS5346 did not show any effect on A1 and A3 ARs in binding or the A2BAR in a cyclic AMP assay at 10 μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A2AAR binding. The FP signal was optimal with 20 nM MRS5346 and 150 μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The Kdvalue of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, Ki values of known AR agonists and antagonists consistently agreed with Ki values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.