Patch Clamping of Single Mammalian Taste Receptor Cell

Abstract

This experiment was carried out to examine the mechanism for the transduction of sweet tastes in mammalian taste receptor cells. We used gerbil taste receptor cells, as the sensitivity to various sweeteners in these animals has been established neuro-physiologically and behaviorally [1,2]. We made both whole cell and cell-attached patch recordings from the single taste cells. After the animal was killed by dislocating the cervical vertebrae, the tongue was removed and 1 mg/ml of elastase was injected into the muscle underlying its dorsal epithelium; the epithelium alone was then peeled off the underlying muscle. When taste receptor cells isolated from the fungiform papillae were incubated in 0.25% trypsin for 20 min at room temperature, a giga ohm seal of the cell membrane patch was easily established. Whole cell recordings, with the pipette containing a standard KC1 solution, showed voltage-activated outward currents and inward currents in the taste cells. Outward K+ currents were induced by depolarizing voltage steps from a holding potential of −60 mV, the threshold of activation being −20 mV. When K+ in the pipette solution was replaced with Cs+, inward rectifier K+ currents were induced by hyperpolarizing voltage steps from a holding potential of −40 mV. The inward rectifier K+ currents were marked at potentials more negative than −120 mV and were suppressed by 10 mM Bat+ added to the bath. When outward currents were blocked by the addition of 10 mM Ba2+ to the bathing solution and by the replacement of K+ in the pipette solution with Cs+, sustained inward Ca2+ currents were observed. The Ca2+ currents were activated at −40mV and reached a peak at +20mV. In whole cell recordings, outward K+ currents were suppressed reversibly by the application of 20 mM Na-saccharin to the bath. In cell-attached patch recordings, a single K+ channel of 85 pS was blocked by 20 mM Na-saccharin, indicating that the blocking of the K+ channel was mediated by a second messenger. In whole cell recordings, 5 mM cpt-cAMP applied to the bath also blocked outward K+ currents. That cAMP is the second messenger has already been proposed by Behe et al. [3] in their experiments in rat taste cells. In the present experiment, we clearly observed Na-saccharin-induced blocking of K+ currents in both whole cell and single channel recordings. Our results support the hypothesis that the signal transduction of sweet tastes in mammalian taste receptor cells is mediated by cAMP-dependent blocking of K+ channels.