The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate

Abstract

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

Figure 1

Effects of the PLC blocker U73122 and PIP₂ on whole-cell currents through TRPM4. (A) Time course of the whole-cell currents after activation by 3 μM Ca²⁺ via the patch pipette. Shown are the currents obtained from voltage ramps at −100 (triangles) and +100 mV (circles), respectively. The IV curves at the right side panel are from the currents indicated by solid circles. Arrows mark the onset of the whole-cell mode. (B) Same experiment as in (A), but now in the presence of 10 μM U73122. Note the complete absence of desensitization and the nearly linear IV curves (right-side panel, current points are marked at the time course). (C) Same experiment as in (A). After desensitization, U73122 was applied to the bath. Note the slow recovery of the current traces (IV curves from ramps as in (A). (D) Whole-cell configuration was established with 3 μM Ca²⁺ and 10 μM diC8-PIP₂ in the pipette. After initial desensitization, the currents completely recovered (traces as the right-hand side from the indicated points).

Figure 2

PIP₂ and U73122 counteract desensitization of TRPM4 in inside-out patches. (A) TRPM4 current measured at −100 and +100 mV using the step protocol in an inside-out patch excised in a solution containing 100 μM Ca²⁺. Note that desensitization reaches a steady-state level. Application of 10 μM PIP₂ reverses the desensitization. (B) Current traces obtained at the time-points indicated in (A). Note the nearly complete disappearance of the time dependence after PIP₂ application. Same scaling as in figure (A). Protocol used to determine the dose dependence of the action of PIP₂ on TRPM4 (C) and resulting dose–response curve (D). The increase in outward current induced by a specific PIP₂ concentration was normalized to that obtained with 50 μM PIP₂. (E) PIP₂ and U73122 applied before patch excision prevent desensitization of TRPM4. (F) Dose–response curve for the effect of U73122 on TRPM4 desensitization. Each data point is from at least four independent measurements. Data points represent the ratio between the steady-state current and the maximal current measured immediately after application of Ca²⁺.

Figure 3

PIP₂ modulates the voltage dependence of TRPM4 in inside-out patches. (A) Current measured in response to the indicated voltage protocol in the absence of PIP₂. (B) Voltage dependence of steady-state and tail current from the experiment shown in panel (A). (C) Voltage dependence of the open probability obtained from normalization of the tail current values to I_max. (D–F) Same as panel (A–C), but now in the presence of 10 μM PIP₂. (G) Activation curves for TRPM4 in control conditions and in the presence of either 10 μM PIP₂ or 10 μM U73122. Each data point is from 2–4 independent measurements. Solid lines represent fits using equation (3). (H) Average values for V_1/2 and z obtained from experiments as in panels (A–F).

Figure 4

PIP₂ increases the Ca²⁺ sensitivity of TRPM4 in inside-out patches. (A) Representative time course of TRPM4 currents at −100 and +100 mV in an inside-out patch, illustrating the Ca²⁺ dependence of TRPM4 activation after desensitization. (B) Original traces from voltage steps obtained from different [Ca²⁺]_i indicated in (A). (C) Same as in (A), but in the presence of 10 μM PIP₂. (D) Original traces from voltage steps obtained from different [Ca²⁺]_i indicated in (C). (E) Dose dependence of TRPM4 activation by Ca²⁺ in the desensitized state and after addition of 10 μM PIP₂. The EC₅₀ value for Ca²⁺ activation changed from 134 μM (control, open circles, n_H=0.9; see also Nilius et al, 2004) to 1.3 μM (n_H=1.0).

Figure 5

Wortmannin and poly-L-lysin attenuate currents through TRPM4 and reversion by PIP₂ in inside-out patches. (A) Time course of whole-cell currents measured as described in Figure 1 (circles +100 mV, triangles −100 mV). Activation of the current was established by 3 μM [Ca²⁺]_i in the patch pipette. (B) Current activation as in (A). Wortmannin (WM, 50 μM, same calibration as in (A)) was included in the patch solution. Note the much smaller currents and the faster desensitization as compared with (A). Preincubation with 50 μM WM for 25 min completely prevented activation of TRPM4 currents with 3 μM [Ca²⁺]_i (data not shown). (C) Pooled data from the maximal current obtained after breaking into the cells with 300 μM [Ca²⁺]_i (supramaximal concentration for TRPM4; Ullrich et al, 2005). Note the decrease in the activated current by WM applied via the pipette and the nearly complete attenuation of the current for 25-min preincubation. (D) From the time courses of current activation with 300 μM [Ca²⁺]_i, the time to 50% desensitization was measured. This time was highly significantly shortened by application of WM into the pipette (t_1/2 values not measurable for 25-min preincubation). (E) Currents measured from inside-out patches (300 μM [Ca²⁺]_i). Before patch excision, cells were incubated for 25 min in 50 μM WM. Note that the first current after excision is very small (see panel F). Application of PIP₂ restored the currents immediately. Typically, washing out of Ca²⁺ resulted in a delayed current decay (circles +100 mV, triangles −100 mV). Traces at the times indicated in the time course are shown at the right-hand side. (F) Averaged current size under control conditions (no WM preincubation, 300 μM [Ca²⁺]_i measured at +100 mV) and after 25-min preincubation with 50 μM WM. (G) Comparison of the first current after excision (preincubation with WM) with the maximal current after 10 μM PIP₂ application (cont: PIP₂ application without WM, currents measured at +100 mV). (H) Time course of TRPM4 desensitization in inside-out patches (300 μM [Ca²⁺]_i). The time course at +100 mV is shown (same step protocol as in (A)). Poly-L-lysin (PLL) is added to the inner side of the membrane in an inside-out patch. PLL (10 μg/ml) already blocked the current completely. Reapplication of 10 μM PIP₂ recovered the current to nearly the same size as the steady-state value. (I) Current traces for the indicated points in the time course shown in (H). (J) Concentration–response curve for the PLL block. Data were fitted by equation (4). The IC₅₀ value is 0.6 μg/ml (n_H=1.1). (K) Recovery from desensitization measured from experiments as shown in (H), in which 10 μM PIP₂ was applied after complete block by PLL. Steady-state current (e.g. trace b in panel H) was obtained from the stationary current divided by the maximal current (I_ss/I_max)). The PIP₂ recovered current was measured by normalizing the maximal recovered current with the maximal current immediately after Ca²⁺ application, I_recovery/I_max. Both values are not statistically significant.

Figure 6

Activation of the M1 receptor inhibits TRPM4 in M1 receptor-expressing CHO cells. (A) Repetitive activation of TRPM4 by 2 μM ION. The IV curves (right) were obtained at the indicated time points (left). (B) Same protocol as in (A), but 100 μM ACh was applied between the first two ION applications. As a consequence, the second application of ION resulted in a much smaller current. A third ION application revealed partial recovery. (C) Averaged data from experiments as in (A) and (B). Current at +100 mV during the second ION application was normalized to that of the first ION application. The normalized current after a 3-min recovery period (recov) is also indicated. (D) Control currents in inside-out patches excised in 300 μM [Ca²⁺]_i. Traces indicated in the time course are shown at the right (the same current calibration). (E) Same as in (D), but now obtained from cells preincubated for 60 s with 100 μM ACh. (F) Summary of the experiments on inside-out patches shown in (D) and (E). Both the peak currents (obtained immediately after patch excision) and steady-state currents were significantly decreased after preincubation with ACh (P<0.05, n=12). Shown are outward currents at +100 mV (columns up) and −100 mV (columns down).

Figure 7

Schematic representation of mutations within putative PIP₂-binding regions of TRPM4. (A) Pairwise alignment of TRP domains of TRPM4 and TRPM5. Identical residues are shown in red within the yellow boxes, homologous residues in black within the green boxes. Note that the positively charged residues are conserved. Residues mutated in this study are marked by asterisks and are identical with the TRP box and TRP domain mutations used by Rohacs et al (2005). (B) Comparison of two putative PH domains in TRPM4 (dotted lines) with the corresponding region in TRPM5. Note the loss of the PH domains in TRPM5. Asterisks with numbers denote TRPM4 residues mutated in this work (for details, see Materials and methods).

Figure 8

Effects of PIP₂ on TRP domain mutants in inside-out patches. (A) Time course showing WT TRPM4 current desensitization at −100 and +100 mV after excision in 300 μM [Ca²⁺]_i (same protocol as in Figure 2), followed by reversal of desensitization upon application of 10 μM PIP₂. To the right, raw current traces obtained at the indicated time points are shown using the same current calibration. (B–D) Same as in panel (A), but now for the indicated TRP domain mutants. (E) Averaged data showing the steady-state current before PIP₂ application (I_ss/I_max) and the current after PIP₂-induced recovery (I_recovery/I_max), both normalized to the maximal current immediately after excision. Note that even the completely desensitizing mutants show unchanged recovery with PIP₂.

Figure 9

Effects of PIP₂ on putative PH domain mutants in inside-out patches. (A) Time course showing current desensitization at −100 and +100 mV for the ΔR/K mutant after excision in 300 μM [Ca²⁺]_i (same protocol as in Figure 2), followed by partial reversal of desensitization upon application of 30 μM PIP₂. To the right, raw current traces obtained at the indicated time points are shown using the same current calibration. PIP₂ (10 μM), which was used in most other experiments, did not induce any recovery of this mutant (n=6, see E). (B) Same as (A), but now for the R1147Q mutant. Note the complete current recovery after application of 10 μM PIP₂. (C) Same as (A), but now for the RKR1147XAAA mutant. Note the small and rapidly desensitizing initial currents. In this case, application of 10 μM PIP₂ resulted in currents that were several-fold larger than the initial peak current (i.e. I_recovery/I_max≫1; see panel E). (D) Same as (A), but now for the TRPM4/cM5 chimeric channel. Note the slow desensitization and the negligible recovery upon application of PIP₂. (E) Averaged data showing the steady-state current before PIP₂ application (I_ss/I_max) and the current after PIP₂-induced recovery (I_recovery/I_max), both normalized to the maximal current immediately after excision. PIP₂ (10 μM) was used, unless noted otherwise.