The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate
2006; Springer Nature; Volume: 25; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7600963
ISSN1460-2075
AutoresBernd Nilius, Frank Mahieu, Jean Prenen, Annelies Janssens, Grzegorz Owsianik, Rudi Vennekens, Thomas Voets,
Tópico(s)Phytochemicals and Antioxidant Activities
ResumoArticle19 January 2006free access The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate Bernd Nilius Corresponding Author Bernd Nilius Department of Physiology, Campus Gasthuisberg Search for more papers by this author Frank Mahieu Frank Mahieu Search for more papers by this author Jean Prenen Jean Prenen Search for more papers by this author Annelies Janssens Annelies Janssens Search for more papers by this author Grzegorz Owsianik Grzegorz Owsianik Search for more papers by this author Rudi Vennekens Rudi Vennekens Search for more papers by this author Thomas Voets Thomas Voets Search for more papers by this author Bernd Nilius Corresponding Author Bernd Nilius Department of Physiology, Campus Gasthuisberg Search for more papers by this author Frank Mahieu Frank Mahieu Search for more papers by this author Jean Prenen Jean Prenen Search for more papers by this author Annelies Janssens Annelies Janssens Search for more papers by this author Grzegorz Owsianik Grzegorz Owsianik Search for more papers by this author Rudi Vennekens Rudi Vennekens Search for more papers by this author Thomas Voets Thomas Voets Search for more papers by this author Author Information Bernd Nilius 1, Frank Mahieu, Jean Prenen, Annelies Janssens, Grzegorz Owsianik, Rudi Vennekens and Thomas Voets 1Department of Physiology, Campus Gasthuisberg *Corresponding author. Laboratory of Physiology, Campus Gasthuisberg, Herestraat 49, KU Leuven, 3000 Leuven, Belgium. Tel.: +32 16 34 5937; Fax: +32 16 34 5991; E-mail: [email protected] The EMBO Journal (2006)25:467-478https://doi.org/10.1038/sj.emboj.7600963 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Phosphoinositides (PI) are ubiquitously used signaling molecules in eukaryotic cells. Phosphatidylinositol 4,5-biphosphate (PIP2) comprises about 3% of the total acidic membrane lipids and more than 99% of the doubly phosphorylated PI in a mammalian cell (McLaughlin et al, 2002). The list of ion channels that are regulated by PIP2 includes inward-rectifier and voltage-gated K+ channels, the two-P domain K+ channels, voltage-gated Ca2+ channels, cyclic nucleotide-gated channels, intracellular Ca2+ ([Ca2+]i) release channels and the epithelial Na+ and Cl− channels (for a recent review, see Suh and Hille, 2005). More recently, it was shown that several members of the transient receptor potential (TRP) channel superfamily are also regulated by PIP2. TRPV1 is tonically inhibited by PIP2 (Chuang et al, 2001; Prescott and Julius, 2003), whereas TRP channel, melastatin subfamily (TRPM)5 (Liu and Liman, 2003), TRPM7 (Runnels et al, 2002), TRPM8 (Liu and Qin, 2005; Rohacs et al, 2005) and TRPV5 (Lee et al, 2005; Rohacs et al, 2005) are all activated in the presence of PIP2. Rohacs et al (2005) proposed a general role for the proximal C-terminal TRP domain in PIP2 regulation of TRPM8 and other PIP2-activated TRP channels, whereas Prescott and Julius (2003) identified a more distal C-terminal region as a crucial determinant of PIP2 inhibition. TRPM4 is a widely expressed member of the melastatin subfamily of TRP channels. It functions as a Ca2+-activated and voltage-dependent monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways (Launay et al, 2002; Hofmann et al, 2003; Nilius et al, 2003). This physiological role was nicely illustrated in T-lymphocytes, where TRPM4 provides a negative feedback on Ca2+ entry, thereby allowing the Ca2+ oscillations that control T-cell cytokine release (Launay et al, 2004). A hallmark feature of TRPM4 is its rapid desensitization to [Ca2+]i (Nilius et al, 2004; Zhang et al, 2005). The mechanisms that underlie this desensitization are poorly understood. It has been shown that the Ca2+ sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation and by binding of calmodulin at the C-terminus (Nilius et al, 2005a). Here we show that PIP2 strongly enhances TRPM4 activity, by increasing the channel's Ca2+ sensitivity and shifting its voltage dependence of activation towards negative potentials. During revision of this article, the group of Liman (Zhang et al, 2005) published comparable observations. In addition, we provide complex mechanistic insights into the PIP2-dependent activation of the channel and present data that suggest a close interaction between TRPM4 and enzymes involved in the PIP2 metabolism. Finally, we demonstrate that the TRP domain is not crucial for the PIP2 effect on TRPM4, and identify positively charged amino acids in a C-terminal pleckstrin homology (PH) domain as important determinants of PIP2 action. Thus, our data provide further evidence that PIP2 is a general regulator of TRP channels, and implicate the C-terminal PH domain of TRPM4 in PIP2 sensing. Results Effect of the phospholipase C inhibitor U73122 and PIP2 on TRPM4 desensitization A typical feature of whole-cell TRPM4 currents activated by high [Ca2+]i is the rapid decay, which reflects a progressive decrease in the Ca2+ sensitivity of the channel (Figure 1A; see also Hofmann et al, 2003; Nilius et al, 2003, 2004, 2005a). We first examined whether Ca2+-dependent activation of a phospholipase C (PLC) leading to PIP2 depletion may underlie desensitization of TRPM4 (as suggested in Rohacs et al, 2005). Addition of the aminosteroid derivative U73122, an inhibitor of receptor-mediated PLC (Bleasdale et al, 1990; Smith et al, 1990), strongly attenuated the desensitization after activation by Ca2+ (Figure 1B). When U73122 was present in the extracellular solution before Ca2+-induced activation, the typical outward rectification of the current was also strongly reduced (Figure 1A and B, right panels). Application of U73122 after maximal current decay led to a significant reversal of the desensitization process (Figure 1C; Irecovery/Imax=0.72±0.13, n=6, measured at +100 mV). U73343, the homolog of U73122 that is ineffective on PLC, did not change desensitization of TRPM4 (data not shown). Furthermore, inclusion of the none-metabolizable PIP2 diC8-PIP2 (from hereon referred to as PIP2) in the intracellular solution resulted in full recovery of the TRPM4 current after the initial decay (Figure 1D; Irecovery/Imax=1.06±0.14, n=8, +100 mV). 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). Download figure Download PowerPoint In inside-out patches, Ca2+-activated TRPM4 currents rapidly decay to a non-zero steady-state level, due to a time-dependent reduction of the channel's Ca2+ sensitivity (Nilius et al, 2004). With 100 μM Ca2+, currents at +100 mV decayed to approximately one-third of the maximal current after excision (Iss/Imax=0.35±0.09, n=5; see Figure 2A). Application of 10 μM PIP2 to the cytosolic side of the inside-out patch resulted in a fast and full recovery of the current (Irecovery/Imax=1.07±0.15, n=5; Figure 2A) accompanied by an apparent loss of the voltage-dependent kinetics (Figure 2B). Recovery from desensitization by application of PIP2 occurred with an EC50 of 5.1 μM (Figure 2C and D), similar to the EC50 reported elsewhere (Zhang et al, 2005). Washout of PIP2 led to slow current decay to a level similar to that obtained for desensitization in the absence of PIP2 (Iss/Imax=0.28±0.11, n=4; compare Figure 2C and E). When 10 μM PIP2 was applied to the bath before patch excision, desensitization was no longer observed (Iss/Imax=0.97±0.09, n=6; Figure 2E). Likewise, blockade of PLC activity using 10 μM U73122 fully prevented desensitization of TRPM4 in inside-out patches (Iss/Imax=0.95±0.07, n=5; Figure 2E), whereas U73343 was ineffective (Iss/Imax=0.32±0.09, n=3). The effect of U73122 on the desensitization process was dose-dependent, with a concentration for half-maximal effect (EC50) of 0.31 μM (Figure 2F). 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+. Download figure Download PowerPoint When after maximal desensitization Ca2+ was removed and subsequently re-added, recovery was never observed. Likewise, U73122 was ineffective on desensitized inside-out patches (data not shown). However, when during the Ca2+-free period Mg-ATP was present at the cytosolic side, currents through TRPM4 recovered to nearly the same size as the maximal current after excision (Nilius et al, 2005a), and this recovery process was further potentiated by U73122 (Supplementary Figure S1). We conclude that Mg-ATP can reverse desensitization of TRPM4 by restoring the PIP2 levels in the membrane patch. Indeed, Mg-ATP is known to activate phosphatidylinositol-4-kinases (PI-4-K), which regenerate PIP2 from PI(4)P (Balla, 2001; Hilgemann et al, 2001). These results also indicate that both PI-4-K and PLC activities remain preserved in TRPM4-containing inside-out patches. To determine whether other PI have a similar effect on TRPM4, we tested seven different PI-phosphates (Supplementary Figure S2). The following potency order was determined in inside-out patches (300 μM [Ca2+]i): PI(4,5)P2>PI (3,4,5)P3=PI(3,5)P2=PI(3,4)P2=PI(5)P>PI(4)P=PI(3)P. Given that PIP2 is both the most potent and the most abundant PI-phosphate, it is likely to represent the physiologically most important regulator of TRPM4. It should be noted here that PIP2 or U73122 did not have any effect on inside-out patches from nontransfected cells or on TRPM4-containing patches in the absence of activating Ca2+, excluding nonspecific actions of these compounds (data not shown). Moreover, we confirmed that 10 μM PIP2 also caused recovery of TRPM4 endogenously expressed in umbilical vein-derived EA cells (Supplementary Figure S3). PIP2 and U73122 shift the voltage and Ca2+ dependence of TRPM4 activation Many regulators of TRPM4 alter the voltage dependence of channel activation (Nilius et al, 2005a). Therefore, we examined the effects of PIP2 and U73122 on the voltage dependence of TRPM4. In the absence of U73122 and PIP2, hyperpolarizing voltage steps from a holding potential of 0 mV caused current deactivation, whereas activation was evident at positive potentials (Figure 3A). Nearly steady-state current–voltage (IV) relationships obtained at the end of 400-ms voltage steps were fitted with a function that combines a linear conductance multiplied by a Boltzmann activation term: 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 Imax. (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 V1/2 and z obtained from experiments as in panels (A–F). Download figure Download PowerPoint where g is the whole-cell conductance, Erev is the reversal potential, V1/2 is the potential for half maximal activation and s is the slope factor. The slope factor s is related to the effective gating charge z of the voltage sensor according to where R is the gas constant, T the absolute temperature and F the Faraday constant. As an alternative way to determine the voltage-dependent gating parameters, tail currents at −100 mV were measured and normalized to the maximal tail current Imax (Figure 3A and B), yielding the voltage dependence of the steady-state open probability of TRPM4 (Figure 3C). Normalized tail currents were fitted using the equation Equivalent values of s and V1/2 were obtained by either method. As described in detail elsewhere, the s for TRPM4 is quite high, indicating that z is small (<1) (Figure 3; Nilius et al, 2005b). Application of 10 μM PIP2 led to an almost complete loss of the time dependence of TRPM4 activation at positive potentials, a dramatic slowing of current deactivation at negative voltages and significant steady-state inward currents (Figure 3D and E). The apparent steady-state open probability was shifted towards negative potentials and was shallower than in the absence of PIP2 (Figure 3F–H). Virtually identical changes in voltage dependence were observed upon application of U73122 (Figure 3E and F and Supplementary Figure S4). As described already in detail (Nilius et al, 2004), the decay of the TRPM4 current in inside-out patches is faster and more complete at low [Ca2+]i, indicating that the Ca2+ sensitivity of the channel decreases after patch excision (Figure 4A). Inward currents are small (Figure 4B). Addition of 10 μM PIP2 to the patch restored the Ca2+ sensitivity. Under persistent PIP2 application, maximal TRPM4 current was already obtained at a [Ca2+]i of 10 μM. Inward currents are large (note the relatively large currents at 0.5 μM [Ca2+]i; Figure 4C and D). We quantified this effect by normalizing the steady-state current to the peak current obtained immediately upon patch excision in the different Ca2+ concentrations. The resulting values were fitted by a dose–response curve of the form: 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, nH=0.9; see also Nilius et al, 2004) to 1.3 μM (nH=1.0). Download figure Download PowerPoint where EC50 represents the concentration for half-maximal TRPM4 activation and nH the Hill coefficient. As shown in Figure 4E, application of 10 μM PIP2 shifted the EC50 value for Ca2+ from 134±21 μM (control) to 1.3±0.2 μM. A similar 10-fold increase in Ca2+ sensitivity was observed in the presence of U73122 (Figure 4E and Supplementary Figure S5). Interestingly, channel closure upon removal of Ca2+ was severely slowed down by PIP2 and U73122. In the absence of these compounds, perfusion with an EGTA-containing Ca2+-free solution led to a virtually immediate inactivation of current (time constant <1 s). In contrast, in the presence of PIP2 or U73122, removal of Ca2+ led to a slow decay of the currents, with exponential time constants in the range of 6–20 s, n=4 (see Supplementary Figure S6). This suggests that PIP2 decreases the rate of Ca2+ unbinding from the channel. Inhibition of TRPM4 by various PIP2-depleting protocols Wortmannin, an inhibitor of PI-4-K, retards the replenishment of PIP2, leading to depletion of the intracellular PIP2 pool (Nakanishi et al, 1995). In the whole-cell mode, inclusion of 50 μM wortmannin in the patch pipette reduced the amplitude of the Ca2+-activated current and led to a faster current decay (Figure 5A–C). Moreover, whole-cell TRPM4 currents were almost fully absent in cells pretreated for 25 min with 50 μM wortmannin (Figure 5D). Next, we tested whether the effect of wortmannin could be reversed by direct application of PIP2. Indeed, after a 25-min pretreatment with 50 μM wortmannin, currents in inside-out patches ([Ca2+]i=300 μM) were strongly decreased (Figure 5E–G), and subsequent application of 10 μM PIP2 led to a strong current increase (Figure 5E–G). 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 (t1/2 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 (nH=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 (Iss/Imax)). The PIP2 recovered current was measured by normalizing the maximal recovered current with the maximal current immediately after Ca2+ application, Irecovery/Imax. Both values are not statistically significant. Download figure Download PowerPoint Poly-L-lysine (PLL) is a positively charged macromolecule that acts as a scavenger of PIP2 (Lopes et al, 2002; Zhang et al, 2003). In inside-out patches, TRPM4 currents were rapidly inhibited when PLL was applied during the steady-state phase (Figure 5H and I). Inhibition by PLL was dose-dependent, and half-maximal inhibition was achieved at a concentration of 0.6 μg/ml (Figure 5J). TRPM4 currents were not restored upon washout of PLL, but subsequent application of PIP2 led to partial reactivation of the channel (Figure 5H and K). Inositol polyphosphate 5-phosphatases (5ptases) are enzymes that remove the phosphate group at the D5 position of PIP2, leading to significant depletion of intracellular PIP2 (Kisseleva et al, 2002). Using HEK cells with tetracyclin-inducible expression of 5ptase IV, we found that induction of this enzyme strongly reduces the peak and steady-state TRPM4 currents in inside-out patches (Supplementary Figure S7). Remarkably, application of PIP2 reactivated TRPM4 currents in only ∼40% of the induced cells, suggesting a high 5ptase activity in the close vicinity of TRPM4 channels. Finally, we tested the effect of hormonal stimulation of PLC-coupled receptors on TRPM4 channel function. For this purpose, we used Chinese hamster ovary (CHO) cells permanently expressing the muscarinic receptor M1 (Buckley et al, 1989) and brief pulses of ionomycin (ION; 2 μM) to repetitively activate TRPM4 currents. In control conditions, a second pulse of ION applied 60–90 s after the first application led to a significant increase of the TRPM4 current at +100 mV (Figure 6A and C). In contrast, activation of the M1 receptor with 100 μM acetylcholine (ACh) after the first ION pulse led to a strong reduction of the current induced by the second ION pulse (Figure 6B and C). Under this condition, a third ION pulse applied 150–180 s after the washout of ACh revealed a partial recovery of the current (Figure 6B and C). 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). Download figure Download PowerPoint We also tested whether M1 activation before patch excision would affect the current size in inside out patches. A 60-s preincubation with 100 μM ACh significantly reduced the first current measured after patch excision in 300 μM [Ca2+]i (Figure 6D–F). Additionally, desensitization of the currents in pretreated cells was faster and more complete then in untreated cells (compare Figure 6D and E). Thus, PLC-coupled receptor activation mimics the effects of PIP2 depletion on TRPM4. The C-terminal PH domain is a putative PIP2 interacting site Rohacs et al (2005) recently reported that mutating positively charged residues in the TRP box and TRP domain of TRPM8 led to a 10–100-fold reduction in the PIP2 sensitivity. Moreover, equivalent mutations in the TRP domain of TRPM5 and TRPV5 increased the sensitivity of these channels to wortmannin, suggestive of a reduced PIP2 sensitivity. They concluded that the TRP domain may be a general PIP2-interacting site in different TRP channels. We investigated whether the corresponding mutations in TRPM4, K1059Q and R1062Q in the TRP box and R1072Q in the TRP domain affect the response to PIP2 in inside-out patches (Figure 7A). In comparison to WT TRPM4, R1062Q and R1072Q displayed a more complete desensitization, with steady-state outward current amplitudes that amounted to less than 5% of the peak current amplitude (Figure 8C–E), whereas K1059Q currents decayed to a similar steady-state level as WT TRPM4 (Figure 8A, B and E). Surprisingly, application of 10 μM PIP2 after maximal desensitization resulted in robust reactivation of all three mutants (Figure 8A–D). The degree of recovery, quantified as Irecovery/Imax, was not significantly different between WT TRPM4 and the three TRP domain mutations (Figure 8E). These data indicate that the TRP box and TRP domain are not the main determinants of PIP2 action, and urged us to search for alternative PIP2 interacting sites in TRPM4. 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). Download figure Download PowerPoint 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 (Iss/Imax) and the current after PIP2-induced recovery (Irecovery/Imax), both normalized to the maximal current immediately after excision. Note that even the completely desensitizing mutants show unchanged recovery with PIP2. Download figure Download PowerPoint The C-terminus of TRPM4 contains two regions of positively charged residues that obey the consensus sequence of PH domains ([R/K]–X3−11–[R/K]–X–[R/K]–[R/K], where X is any amino acid; see http://us.expasy.org/prosite/; Figure 7B), which are known as PIP2 interaction sites (Harlan et al, 1994). The first, most proximal putative PH domain, R1136ARDKR1141, was previously shown to determine modulation of TRPM4 by decavanadate (Nilius et al, 2004). Neutralization of all four basic residues in this region resulted in a mutant channel (termed ΔR/K) that exhibited fast and complete desensitization in inside-out patches (Figure 9
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