Abstract
TRPP3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a Ca2+-activated channel permeable to Ca2+, Na+, and K+. TRPP3 has been implicated in sour tasting in bipolar cells of tongue and in regulation of pH-sensitive action potential in spinal cord neurons. TRPP3 is also present in excitable and nonexcitable cells of other tissues, including retina, brain, heart, testis, and kidney, with unknown functions. In this study, we examined the functional modulation of TRPP3 channel by amiloride and its analogs, known to inhibit several ion channels and transporters and respond to all taste stimuli, using Xenopus laevis oocyte expression, electrophysiology, and radiotracer measurements. We found that amiloride and its analogs inhibit TRPP3 channel activities with different affinities. Radiolabeled 45Ca2+ uptake showed that TRPP3-mediated Ca2+ transport was inhibited by amiloride, phenamil, benzamil, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Two-microelectrode voltage clamp experiments revealed that TRPP3-mediated Ca2+-activated currents are substantially inhibited by amiloride analogs, in an order of potency of phenamil > benzamil > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 μM, respectively. The inhibition potency positively correlated with the size of inhibitors. Using cell-attached patch clamping, we showed that the amiloride analogs decrease the open probability and mean open time but have no effect on single-channel conductance. Study of inhibition by phenamil in the presence of previously reported inhibitor tetrapentylammonium indicates that amiloride and organic cation inhibitors compete for binding the same site on TRPP3. TRPP3 may contribute to previously reported in vivo amiloride-sensitive cation transport.
Footnotes
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This work was supported by the Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Kidney Foundation of Canada (to X.-Z.C.). X.-Z.C. is an AHFMR Senior Scholar. X.-Q.D. is the recipient of an AHFMR Studentship.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.037150.
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ABBREVIATIONS: TRP, transient receptor potential; ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; EIPA, 5-(N-ethyl-N-isopropyl) amiloride; ENaC, epithelial sodium channel; MOT, mean open time; NPo, open probability; ANOVA, analysis of variance; TPeA, tetrapentylammonium; I-V, current-voltage relationships; TRPP2: transient receptor potential polycystin-2; TRPP3: transient receptor potential polycystin-L; TRPP5: transient receptor potential polycystin-2L2.
- Received April 16, 2007.
- Accepted September 4, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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