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. 2003 Dec 9;100(25):15160-5.
doi: 10.1073/pnas.2334159100. Epub 2003 Dec 1.

Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5

Dan Liu  1 , Emily R Liman
Affiliations

Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5

Dan Liu et al. Proc Natl Acad Sci U S A. .

Abstract

The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C beta2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is not known. Here we show that heterologously expressed TRPM5 forms a cation channel that is directly activated by micromolar concentrations of intracellular Ca2+ (K1/2 = 21 microM). Sustained exposure to Ca2+ desensitizes TRPM5 channels, but PIP2 reverses desensitization, partially restoring channel activity. Whole-cell TRPM5 currents can be activated by intracellular Ca2+ and show strong outward rectification because of voltage-sensitive gating of the channels. TRPM5 channels are nonselective among monovalent cations and not detectably permeable to divalent cations. We propose that the regulation of TRPM5 by Ca2+ mediates sensory activation in the taste system.

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Figures

Fig. 1.
Fig. 1.
TRPM5 is activated by micromolar concentrations of Ca2+.(a) Activation by 40 μM Ca2+ of an inward current in a patch excised from a TRPM5-transfected CHO-K1 cell (Vm =-80 mV). Neither 10 μMIP3 nor 100 μM OAG elicited a current in the same patch. (b) Single-channel recording at different voltages in response to 12 μM Ca2+ (Left). The I–V relation of the same patch (Right) yields a slope conductance of 16 pS. No channel activity was seen in this patch in the absence of Ca2+.(c) Responses of TRPM5 channels to increasing concentrations of Ca2+. Note the desensitization of the channels in response to repeated application of the stimulus protocol. (d) Dose–response relations from patches (Vm = -80 mV) before (filled circles) or after (open circles) desensitization (mean ± SEM, n = 3 patches; data were normalized to the peak current in each patch). Fits are to the Hill equation with K1/2 = 21 μM, n = 2.4, and Vmax = 1.05 before desensitization (solid line) and K1/2 = 77 μM, n = 2.4, and Vmax = 0.23 after desensitization (dashed line; note this fit was obtained by holding n constant, but allowing Vmax and K1/2 to vary). The dotted line shows the predicted relationship if only the maximum current were to decline (simulation with K1/2 = 21 μM, n = 2.4, and Vmax = 0.23), illustrating that with desensitization the dose–response relationship shows a shift in sensitivity as well as a decrease in the maximal current activated.
Fig. 2.
Fig. 2.
Desensitization of TPRM5 is Ca2+-dependent. (a) Responses in an excised patch (-80 mV) to repeated applications of Ca2+. Note that the current declined gradually in response to repeated exposures to 40 μM Ca2+ but declined dramatically in response to 500 μM Ca2+. (b) Response in patches to prolonged exposure to Ca2+ (-80 mV). Single exponential fits are shown in red. (c) The rate of decay of the current (1/τ) is linearly related to Ca2+ concentration, over the range measured, indicating that desensitization is Ca2+-dependent (mean ± SEM, n = 5 patches for each data point).
Fig. 3.
Fig. 3.
PIP2 partially restores TRPM5 channel activity after desensitization. (a) Responses to 12 and 40 μM Ca2+ in the presence and absence of 20 μM PIP2 before and after desensitization. Desensitization was induced by a 30-s exposure to 40 μM Ca2+ (Vm = -80 mV). PIP2 enhances the current in response to Ca2+ after, but not before, desensitization. (b) Enhancement of the current in response to 40 μM Ca2+ by PIP2 before (n = 6) and after desensitization (n = 8). Current amplitudes are the averaged peak magnitude recorded within 2 s from the start of Ca2+ exposure. The asterisk indicates that the difference between the enhancement of control and desensitized currents was significant at P < 0.05. (c) Dose–response relations before desensitization (open triangles), after desensitization (filled circles), and after desensitization in the presence of 10 μM PIP2 (open circles) (mean ± SEM, n = 3 patches). In these experiments, PIP2 was present before and during a 6-s application of Ca2+. Currents are normalized to the maximum current obtained in each patch. Solid lines are the same fits as in Fig. 1d. The dashed line shows the expected relationship if 25% of the current recovered full sensitivity. The data could not be well fitted by assuming that either only the maximum current or the sensitivity was restored by PIP2.
Fig. 4.
Fig. 4.
Electrophysiological properties of TRPM5 expressed in HEK-293 M1 cells. (a) Application of the Ca2+ ionophore A23187 (20 μM) in the presence of 2 mM extracellular Ca2+ in perforated-patch recording mode elicits a large outwardly rectifying current. (Inset) The current in response to a ramp depolarization (1 V/s) at the times indicated. (b) Application of ACh (100 μM) induced a transient current in HEK-293 M1 cells cotransfected with TRPM5 and G16z44. (Inset) The current in response to a ramp depolarization (0.44 V/s) at the times indicated. The pipette solution contained no Ca2+ buffer. (c) In whole-cell recording mode, 40 μM Ca2+ in the pipette elicited a large rectifying current. Recording began shortly after break into the whole-cell mode. (Inset) The current in response to a ramp depolarization (1 V/s). (d) Currents in response to a family of step depolarizations and the resulting I–V relationship for the peak current at each voltage. Steps are to 0–100 mV from a holding potential of -80 mV with repolarization to -50 mV. Note the prominent relaxation of the current upon depolarization, consistent with voltage-dependent gating of the channels. A small fraction of the current showed a fast increase, indicating that there is low, but nonzero, probability of opening at the resting potential (-80 mV).
Fig. 5.
Fig. 5.
A model for taste transduction. Binding of taste stimuli to G protein-coupled taste receptors (R) leads to dissociation of the heterotrimeric G protein. βγ subunits of the G protein activate PLCβ2, which in turn hydrolyzes PIP2 into DAG and IP3. IP3 activates IP3 receptors, which release Ca2+ from intracellular stores. Intracellular Ca2+ opens TRPM5 channels, leading to an influx of Na+ and depolarization of the cell. Note that TRPM5 is not permeable to Ca2+ and, therefore, there is no positive feedback loop.
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