Background and purpose: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. Experimental approach: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. Key results: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC 50 in the range of 2 Â 10 À5 M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC 50 . Channel inhibition was voltage independent. The IC 50 determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. Conclusions and implications: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.

In the disease cystic fibrosis (CF), the most common mutation delF508 results in endoplasmic reticulum retention of misfolded CF gene proteins (CFTR). We show that the a-1,2-glucosidase inhibitor miglustat (N-butyldeoxynojirimycin, NB-DNJ) prevents delF508-CFTR/calnexin interaction and restores cAMP-activated chloride current in epithelial CF cells. Moreover, miglustat rescues a mature and functional delF508-CFTR in the intestinal crypts of ileal mucosa from delF508 mice. Since miglustat is an orally active orphan drug (Zavesca Ò ) prescribed for the treatment of Gaucher disease, our findings provide the basis for future clinical evaluation of miglustat in CF patients.

Chloride (Cl -) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl -channels in aortic VSMC from Cftr +/+ and Cftr -/-mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr +/+ . On the contrary, in cultured VSMC from Cftr -/-mice these agonists have no effect, showing that CFTR is the dominant Cl -channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca 2+ -dependent Cl -channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTR inh -172, glibenclamide and diphenylamine-2,2 -dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr -/-mice became significantly more constricted (∼2-fold) than that of Cftr +/+ mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr +/+ mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr -/-mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl -channel on vascular reactivity.