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Inhibition of TRPP3 Channel by Amiloride and Analogs

Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada

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.

Address correspondence to: Dr. Xing-Zhen Chen, 729 Medical Sciences Building, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. E-mail: xzchen{at}ualberta.ca

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