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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Cell. Author manuscript; available in PMC Feb 23, 2008.
Published in final edited form as:
PMCID: PMC1855209

Integration of Phosphoinositide and Calmodulin Mediated Regulation of TRPC6


Multiple TRP channels are regulated by phosphoinositides (PIs). However, it is not known whether PIs bind directly to TRP channels. Furthermore, the mechanisms through which PIs regulate TRP channels are obscure. To analyze the role of PI/TRP interactions, we used a biochemical approach, focusing on TRPC6. TRPC6 bound directly to PIs, and with highest potency to phosphatidylinositol 3,4,5-trisphosphate (PIP3). We found that PIP3 binding disrupted the association of calmodulin (CaM) with TRPC6. We identified the PIP3 binding site and found that mutations that increased or decreased the affinity of the PIP3/TRPC6 interaction enhanced or reduced the TRPC6-dependent current respectively. PI mediated disruption of CaM binding appears to be a theme that applies to other TRP channels, such as TRPV1, as well as to the voltage-gated channels, KCNQ1 and Cav1.2. We propose that regulation of CaM binding by PIs provides a mode for integration of channel regulation by Ca2+ and PIs.


Phosphoinositides (PIs), such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), profoundly affect the activities of an array of ion channels ranging from voltage-gated K+ channels to voltage-gated Ca2+ channels, cyclic nucleotide gated channels, ENaC and others (reviewed in Suh and Hille, 2005). The effects of PIP2/PIP3 on ion channel activity can be quite different. While the activities of many channels are promoted by PIs, in other instances they are inhibited.

During the last few years, the theme of PI-mediated regulation of ion channels has been extended to members of the TRP superfamily of cation channels (reviewed in Montell, 2005). These include the capsaicin/heat activated channel, TRPV1, which displays a reduced sensitivity to capsaicin and higher temperature threshold in the presence of PIP2 (Prescott and Julius, 2003). The Drosophila TRPL channel has also been proposed to be negatively regulated by PIP2 (Estacion et al., 2001). However, in contrast to these effects, a more common effect of PIs on TRP channels is that they enhance activity, either as a result of contributing to activation or decreasing desensitization. Such effects of PIP2 or PIP3 have been reported for four TRPM channels (TRPM4, TRPM5, TRPM7 and TRPM8), two TRPV channels (TRPV2 and TRPV5) and TRPC6 (Runnels et al., 2002; Liu and Liman, 2003; Tseng et al., 2004; Lee et al., 2005; Liu and Qin, 2005; Rohács et al., 2005; Zhang et al., 2005; Nilius et al., 2006; Penna et al., 2006).

Despite extensive biophysical analyses of the consequences of PIs on TRP channel activity, there is a lack of biochemical studies concerned with the underlying mechanisms. In particular, it is not known whether TRP channels bind directly to PIs, although mutations in putative PI binding sites in several TRP channels prevented the effects of the PIs (Prescott and Julius, 2003; Rohács et al., 2005; Nilius et al., 2006). If TRPs bind PIs directly, the question arises as to how PI binding might affect TRP channel activity.

To address the mechanism of PI-mediated regulation of TRP channels, we used a biochemical approach, focusing on TRPC6. This channel is expressed in many cell types, including sensory receptor cells (Boulay et al., 1997; Hofmann et al., 1999; Buniel et al., 2003; Elsaesser et al., 2005; Warren et al., 2006). Moreover, mutations in this channel have been shown recently to underlie a kidney disease, referred to as autosomal dominant segmental glomerulosclerosis (Reiser et al., 2005; Winn et al., 2005). In rodents, TRPC6 appears to contribute to pathologic cardiac remodeling (Kuwahara et al., 2006; Onohara et al., 2006) and has an important role in the control of vascular smooth muscle tone (Dietrich et al., 2005).

TRPC6 activity is subject to regulation by calmodulin (CaM) and PIP3. The initial activation of this channel is dependent on stimulation of phospholipase C and production of diacylglycerol (Hofmann et al., 1999). In addition to being augmented by PIP3, TRPC6 binds directly to CaM (Boulay, 2002; Tseng et al., 2004; Zhu, 2005).

In the current report, we show that TRPC6 binds directly to PIs, and with highest affinity to PIP3. Most importantly, we found that addition of PIs disrupts the CaM/TRPC6 interaction. A mutation in TRPC6 that decreases the potency of PIP3-induced disruption of CaM binding, decreases the initial current amplitude. Conversely, an amino acid substitution that enhances the efficacy through which PIP3 competes for CaM binding, increases the TRPC6 current. We found that PIs disrupted CaM binding to many ion channels. These include TRPC1, TRPC7, TRPV1 and two voltage-gated channels, KCNQ1 and Cav1.2. We propose that reduction of CaM binding by PIs is a previously unrecognized mode for coincidence detection of Ca2+ and PI signals, which applies broadly to many classes of ion channels.


PI/IPs bind directly to TRPC6

To test whether TRPC6 associates directly with PIs, we focused on the C-terminal region of TRPC6, as the corresponding region has been implicated in PI regulation in other TRP channels (Prescott and Julius, 2003; Rohács et al., 2005; Nilius et al., 2006). Therefore, we expressed and purified a maltose binding protein (MBP) fused to the entire region C-terminal to the sixth transmembrane segment in TRPC6 (residues 728–931). We used MBP-TRPC6-C to probe PIs immobilized on a nitrocellulose membrane (PIP Strips) and found that the fusion protein, but not MBP alone bound to a variety of PIs (Figure 1A). These data indicated that TRPC6 interacted directly with PIs.

Figure 1
Binding of IP6 and PIs to TRPC6-C. (A) MBP-TRPC6-C (residues 728–931) bound to PIs immobilized on membranes. PIP Strips (Echelon Bioscience) were probed with MBP or MBP-TRPC6-C, followed by rabbit anti-MBP antibodies and horseradish peroxidase-coupled ...

To more precisely quantify the binding characteristics of TRPC6-C to the hydrophilic PI head group, we used IP6, since it is available as a labeled derivative and is soluble in an aqueous solution. We combined [3H]IP6 and purified MBP-TRPC6-C, precipitated the fusion protein and determined the level of radioactivity that was retained in the pellet. Consistent with the filter binding assay, MBP-TRPC6-C bound [3H]IP6 (Figure 1B). To determine the binding specificity, we performed competition assays using [3H]IP6 in combination with different concentrations of unlabeled PIs or IP6. IP6 and PI(3,4,5)P3 were the most effective in competing [3H]IP6, followed by PI(4,5)P2.(Figures 1C and D). Evidence that PIs were more potent than IP6 for binding to TRPC6 is presented below.

Overlap of calmodulin and PI/IP binding domains

To identify the PI binding site in the TRPC6 C-terminal region, we generated a series of MBP-TRPC6 C-terminal fusion proteins (Figure 2A), and assayed [3H]IP6 binding as described above. We found that a region including residues 842–868 was responsible for IP6 binding (Figures 2A and B). This site overlaps with the previously described CaM binding domain in mouse TRPC6 (residues 838–872) (Zhu, 2005). Consistent with these data, we found that the same fragment that bound PIs also associated with CaM and this interaction was Ca2+ dependent (see below).

Figure 2
PI and CaM binding domains (CBDs) overlapped in the TRPC6 C-terminus. (A) Schematic of C-terminal regions of TRPC6 (C6-C) fused to MBP. The TRP domain and CBD are indicated. The residues in TRPC6 included in the fusions are indicated. The sixth transmembrane ...

To map the PI/IP and CaM binding sites further, we changed positively charged residues (lysine or arginine) to glutamine, since mutations in positively charged residues could potentially disrupt PI and CaM binding. We found that a triple substitution of arginine 853, lysine 860 and arginine 861 (Figure 2C; R853Q/K860Q/R861Q) significantly reduced binding to IP6 (Figure 2D; 24.9 ±4.1% of wild-type binding). Mutation of arginine 853 alone (R853Q) also had a large impact on IP6 binding (36.7 ±4.4% of wild-type binding), while the single K860Q or R861Q substitutions resulted in relatively small but reproducible decreases (Figure 2D; 85.9 ±2.8% and 76.5 ±3.2% of wild-type binding, respectively). By contrast, substitution of arginine 865 (R865Q) had no affect on PI binding (97.3 ±13.7% of wild-type binding).

The triple amino acid substitution R853Q/K860Q/R861Q, which significantly decreased PI binding also resulted in a large reduction in CaM binding (3.7 ±0.5% of wild-type binding; Figures 3A–C). Nevertheless, it was possible to identify mutations that had differential effects on PI/IP6 and CaM binding. Both R853Q and R865Q resulted in similar ~30–35% reductions in CaM binding (Figures 3A–C). However, the effects of these mutations on IP6 binding were quite different, as described above (summarized in Figure 3A). While the R853Q substitution resulted in a decrease in IP6 binding, the R865Q substitution had no impact on IP6 binding (Figures 2D and and3A).3A). These data demonstrate that the PI and CaM binding sites overlap (PCaM domain), but are not identical.

Figure 3
Disruption of CaM binding to TRPC6-C by PIs and IP6. (A) Summary of relative CaM and IP6 binding to wild-type MBP-TRPC6-C and derivatives. The relative CaM binding were based on the data in Figures 3B and C. The [3H]IP6 binding was based on Figure 2D ...

Regulation of calmodulin binding by PIs

The preceding observations raise the possibility that PI/IPs and CaM might compete for a common binding site on TRPC6. If so, then addition of PI/IP6 might interfere with CaM binding. To test this possibility, we performed CaM-Sepharose pull-down assays with MBP-TRPC6-C in the presence and absence of various concentrations of IP6, PI, PIP2 and PIP3. We found that PIs disrupted CaM binding (PDC) to TRPC6, and PIP3 was the most potent PI (PDC50=1.2 μM; Figures 3A, D and E). PIP2 was also effective in reducing CaM binding, but less so than PIP3 (PDC50=8.2 μM) (Figures 3A, D and E). IP6 was less potent in competing CaM to TRPC6 (>20 μM), consistent with the conclusion that PIP3 or PIP2 rather than IP6 were key ligands that regulate CaM binding (Figures 3A, D and E).

If the PI-induced reduction in CaM binding operated through the TRPC6 PCaM domain, then the competition should be diminished by the single amino acid substitution that caused a three-fold reduction in PI binding (R853Q; Figure 3A). We found that the R853Q mutation caused nearly a 10-fold decrease in competition by PIP3 (PDC50 = 11.0 μM; Figures 3A and D). Next, we tested the effects of R865Q, which did not decrease PI binding, but caused a 35% reduction in CaM binding. This latter mutation might increase the potency of the PDC. Consistent with this proposal, we found that PIP3 was four-fold more effective in competing CaM binding from R865Q than from wild-type TRPC6 (PDC50 = 0.3 μM; Figures 3A and D). These results further support the conclusion that PIs inhibit the interaction of CaM with TRPC6 through overlapping binding sites in the PCaM domain.

Interactions of PIs and calmodulin regulate TRPC6

To test a role of PI-regulated CaM binding for TRPC6 channel function, we expressed three mutant derivatives of human TRPC6 in HEK293T cells. We examined the whole-cell currents following activation of the H1 histamine receptor, since addition of histamine to mock transfected cells did not elicit discernible currents (Hofmann et al., 2003). One of the TRPC6 derivatives analyzed contained the R853Q change (TRPC6R853Q) as this substitution reduced the potency of the PDC (Figure 3A). We also examined the currents resulting from expression of TRPC6R865Q since the R865Q change resulted in an increase in the potency of the PDC (Figure 3A). Finally, we analyzed a TRPC6 derivative containing the triple substitution, R853Q/K860Q/R861Q (TRPC63Q), since these changes impaired both PI and CaM binding (Figure 2D and and3A3A).

The effects of the R853Q and R865Q mutations were opposite. The initial TRPC6R853Q-induced currents were reduced three-fold relative to wild-type (Figures 4A–D; 0.31 ±0.07 nA, n=12 and 0.91 ±0.16 nA, n=13 respectively). No shift in the reversal potential was observed (Figure 4B). In cells expressing TRPC6R865Q there were two phases to the response. In contrast to the TRPC6R853Q-induced currents, the initial histamine-induced currents in TRPC6R865Q were increased >two-fold relative to wild-type (Figures 4A–C and E; 1.92 ±0.44 nA, n=17). Moreover, the activation and inactivation of this initial current were more rapid than in cells expressing wild-type TRPC6. The ensuing amplitude and kinetics of deactivation of the remaining current were similar to wild-type. Similarly, larger currents were observed in cells expressing TRPC63Q (Figures 4A–C and F; 1.86 ±0.32 nA, n=14). Since both PI and CaM binding are disrupted in TRPC63Q and PI binding is similar to wild-type TRPC6 in TRPC6R865Q, the similarities in currents would not appear to be due to defects in PI binding. Rather, the increased initial current would appear to result from disruption of CaM binding. Consistent with this proposal, PIP3 disrupted the TRPC6/CaM interaction with higher potency in TRPC6R865Q than in wild-type TRPC6 (Figure 3A). TRPC6R853Q and TRPC6R865Q also displayed lower and higher activity than wild-type TRPC6 respectively, when the cells were stimulated with the DAG analog, OAG (data not shown).

Figure 4
Effects of Ca2+ buffering and CaM overexpression on TRPC6-dependent peak current amplitudes. (A) Average initial current amplitudes of TRPC6 derivatives under three different conditions. Grey bars (middle bars in each set) indicate the average initial ...

Membrane localization of wild-type and mutant TRPC6 channels

The changes in initial currents were not due to increases or decreases in surface expression of TRPC6. Using confocal microscopy, the YFP labeling was similar in cells expressing wild-type TRPC6-YFP, and the mutant derivatives: TRPC63Q, TRPC6R865Q and TRPC6R853Q (Figure S1A). To compare the levels of cell surface channels quantitatively, we performed surface biotinylation experiments. We prepared extracts from cells, before and after histamine stimulation, which expressed either wild-type TRPC6 or the mutant derivatives that display larger (TRPC6R865Q and TRPC63Q) or smaller current amplitudes (TRPC6R853Q). To provide a positive control for surface expression, we used mCD8-YFP, since the concentration of this cell surface protein should not be altered by histamine stimulation. Indeed, the concentration of biotinylated mCD8-YFP was unaffected by histamine stimulation (Figure S1B). In those cases in which some differences were observed (Figure S1B, upper panel), they were due to comparable variations in sample loading (Figure S1B, lower panel). A GFP derivative (Venus), which is a cytosolic protein (Nagai et al., 2002), was not biotinylated.

Of importance here, stimulation with histamine had no impact on surface biotinylation of TRPC6. In cells expressing TRPC6R853Q and which displayed smaller histamine induced currents, we did not observe a lower level of surface biotinylation (Figure S1B). Conversely, the levels of biotinylated TRPC6 were not increased by histamine stimulation of cells expressing either TRPC6R865Q or TRPC63Q, which displayed larger initial current amplitudes (Figure S1B). Therefore, in no case did we detect histamine-induced changes in cell surface expression and there was no correlation between the levels of cell surface TRPC6 and the initial current amplitudes.

Ca2+ and calmodulin suppress the TRPC6 current through the PCaM domain

To test further that PIP3 promotes the histamine-induced currents, by disrupting the TRPC6/CaM interaction, we examined the effects on the current amplitudes by buffering intracellular Ca2+. CaM binding to TRPC6 is dependent on Ca2+ (Boulay, 2002); therefore, buffering of Ca2+ should negate the effects of mutations in TRPC6 that affect the potency of the PDC. To test this proposal, we buffered intracellular Ca2+ with BAPTA and compared the initial current amplitudes in cells expressing either the wild-type or mutant TRPC6 channels. As described above, in the absence of Ca2+ buffering, the histamine-induced current amplitudes varied depending on the potency of the PDC50: the current was largest in cells expressing TRPC6R865Q (PIP3 PDC50 was 0.3 μM), intermediate for wild-type TRPC6 (PIP3 PDC50 was 1.2 μM) and smallest in cell expressing TRPC6R853Q (PIP3 PDC50 was 11.0 μM; Figure 3).

To assess the role of Ca2+ on the regulation of TRPC6, we buffered intracellular Ca2+ by addition of 20 mM BAPTA to the pipette solution. We found that in the presence of BAPTA, the initial histamine-induced current amplitudes were similar in cells expressing wild-type TRPC6, or any of the mutant TRPC6 channels (black bars in Figure 4A and Figure S2). In cells expressing TRPC6 and TRPC6R853Q, buffering of intracellular Ca2+ elicited ~two- and ~six-fold increases in TRPC6-induced currents respectively, relative to unbuffered cells (Figure 4A). The observation that the current amplitude increased most dramatically in buffered cells expressing TRPC6R853Q, was consistent with the finding that PIP3 is ten-fold less potent at dissociating CaM from TRPC6R853Q, than from wild-type TRPC6. In the cases of cells expressing TRPC6R865Q or TRPC63Q, the current amplitudes were not changed significantly by buffering intracellular Ca2+ (Figure 4A). These latter data are also consistent with the conclusion that PIP3 promotes the dissociation of CaM, since the TRPC6R865Q/CaM interaction is potently disrupted by PIP3, even in the presence of Ca2+ (Figure 3A). BAPTA had little effect on the current amplitude in TRPC63Q, since the mutations in this TRPC6 isoform nearly eliminated CaM binding (Figure 3A).

As a complementary approach to disrupting CaM binding with BAPTA, we assessed the effect on the wild-type and mutant TRPC6 channel currents, by increasing the intracellular CaM concentration. Introduction of a vector (pcDNA3-CaM-YFP) that encoded CaM suppressed the wild-type TRPC6 current (Figure 4A). However, introduction of pcDNA3-CaM-YFP did not cause significant suppression of the histamine-induced current in cells expressing the TRPC63Q isoform, which binds little if any CaM (Figure 4A). These results provide further support to the conclusion that Ca2+/CaM is involved in channel inhibition, and that the primary CaM binding site is disrupted by the mutations in the PCaM domain in TRPC63Q. Introduction of pcDNA3-CaM-YFP did not affect the TRPC6R853Q-induced currents significantly. Moreover, the average current amplitude of TRPC6R853Q, without pcDNA3-CaM-YFP, was similar to that of wild-type TRPC6 after CaM overexpression (Figure 4A). These data suggest that maximal CaM-induced inhibition was achieved with TRPC6R853Q, even without CaM overexpression, due to the reduced potency of the PDC. In addition, CaM overexpression did not cause a statistically significant decrease in the TRPC6R865Q-induced currents (Figure 4A). This suggests that introduction of additional CaM was insufficient to overcome the increased potency of the PDC50 in TRPC6R865Q. The combination of the effects resulting from buffering Ca2+ and overexpressing CaM further support the conclusion that CaM binding to the PCaM domain in TRPC6 is important for the Ca2+/CaM mediated suppression of TRPC6 activity.

PIP3 “sponge” at the plasma membrane reduces wild-type TRPC6 currents

If PIP3 functions to disrupt CaM from TRPC6, leading to potentiation of TRPC6-dependent currents, then a reduction of PIP3 from the plasma membrane might reduce the histamine-induced currents. To test this proposal, we expressed a AKT-PH domain which binds predominately to PIP3 (James et al., 1996) and therefore can be used as “PIP3 sponge” to reduce PIP3 levels (Sidhu et al., 2005). We found that AKT-PH-GFP was primarily detected at the cell cortex, consistent with plasma membrane localization, while a mutant AKT-PHR25C-GFP (Franke et al., 1997) was exclusively detected in the cytosol (Figure 5A). These results indicate that HEK293T cells have PIP3 at the plasma membrane, which could potentially affect TRPC6 currents. Therefore, we tested whether overexpression of the AKT-PH domain suppressed TRPC6 currents. Introduction of the mutant AKT-PHR25C domain, which does not bind PIP3, has no impact on the histamine-induced current amplitudes in cells expressing either wild-type TRPC6, or the mutant isoforms (compare Figures 4 and 5B–F). However, the wild-type TRPC6-induced currents were decreased by overexpressing the AKT-PH domain (Figures 5B and C). The current amplitudes of TRPC63Q were not altered by overexpressing AKT-PH compared to those of AKT-PHR25C overexpression, consistent with the observation that this isoform did not bind CaM. We did not observe a further reduction in current amplitude in cells expressing TRPC6R853Q, presumably since the current was already reduced greatly due to the decreased potency of the PDC50 resulting from the R853Q mutation (Figure 3A). The slight reduction in the TRPC6R865Q, current was not statistically significant. In conclusion, the reduction in the wild-type TRPC6 current amplitude by AKT-PH, but not by the AKT-PHR25C derivative, provide further support for a role for PIP3 in potentiating the amplitude of the histamine-induced currents.

Figure 5
Suppression of the wild-type TRPC6-dependent current by expression of AKT-PH, but not by expression of AKT-PHR25C, which does not bind PIP3. (A) AKT-PH-GFP localized to the cortex of unstimulated HEK293T cells. pcDNA-AKT-PH-GFP or pcDNA-AKT-PHR25C-GFP ...

Other TRPCs and TRPV1 bind phosphoinositides

All TRPC channels appear to bind CaM (reviewed in Zhu, 2005) raising the possibility that PI-mediated regulation of CaM binding occurs in additional TRPC proteins. To address this possibility, we first tested whether TRPC channels other than TRPC6 bind PIs/IP6. We generated MBP fusions proteins, which included the C-termini of TRPC1, TRPC5 or TRPC7 and performed IP6 binding assays. All TRPC channels tested bound IP6 and the strongest binding was observed for TRPC1 (Figure 6A). To examine these interactions further, we probed PIP Strips with the MBP-TRPC C-terminal fusions. All of the fusion proteins, but not the MBP alone, bound to PIs immobilized on the membranes (Figures 1A and and6B6B).

Figure 6
PI/IPs binding to the C-terminal regions of multiple TRP channels. (A) [3H]IP6 binding to the C-terminal regions of TRP channels fused to MBP: TRPC1 (C1-C; human residues 612–759), TRPC5 (C5-C; mouse residues 625–975), TRPC6 (C6-C; human ...

TRPV1 is regulated by PIs, although direct binding assays have not been reported (Prescott and Julius, 2003; Liu et al., 2005). As was observed with the MBP-TRPC fusion proteins, the C-terminal region of TRPV1 bound IP6 (Figure 6A). Therefore, the C-termini of at least four TRPCs (TRPC1-C, TRPC5-C, TRPC6-C and TRPC7-C) and TRPV1 bind directly to PIs.

Common role for PIs in inhibiting association of CaM to ion channels

The finding that multiple TRPC proteins bind to CaM and PIs raise the possibility that regulation of CaM binding by PIs is a theme common among TRPC proteins. To address this hypothesis, we used the MBP-TRPC-C fusion proteins described above. We found that CaM was displaced by 20 μM PIP2 and PIP3 in TRPC1-C, TRPC7-C (Figure 7). The same concentration of IP6 was ineffective in disrupting CaM binding to TRPC1 and TRPC7. Neither the PIs nor IP6 displaced CaM from TRPC5-C (Figure 7). Thus, PIP2 and PIP3 inhibit CaM binding to several, but not all TRPC proteins. To test whether PI-mediated regulation of CaM binding is a phenomenon that extends to TRP channels other than TRPCs, we examined MBP-TRPV1-C. We found that CaM binding to MBP-TRPV1-C was reduced in the presence of IP6, PIP2 or PIP3 (Figures 7), and was most pronounced in the presence of PIP3.

Figure 7
PI/IPs disrupt CaM binding to multiple types of channels. (A) Interference of channel binding to CaM-Sepharose by IP6/PIs. The experiments were conducted using MBP fused to the C-terminal domains of either TRPC1, TRPC7, TRPV1, KCNQ1 or Cav1.2. The same ...

We also tested whether CaM binding to voltage-gated channels was regulated by PIs. The voltage-gated K+ channel KCNQ1 contains two CaM binding domains located within the C-terminal region of the channel (Delmas and Brown, 2005). This channel also binds to PIP2 in a region overlapping with one of the CaM binding domains (Delmas and Brown, 2005). To test whether CaM binding to KCNQ1 was dissociated by PIs, we fused each of the two C-terminal KCNQ1 CaM binding regions (CBS1 and CBS2) to MBP and performed competition assays. We found that CaM binding to both CBS1 and CBS2 was reduced by PIs and that PIP3 was the most potent at disrupting this interaction (Figure 7). CaM binding to voltage-gated Ca2+ channels is also potentially regulated by PIs since such channels are regulated by both CaM and PIs (reviewed in Wu et al., 2002; Halling et al., 2006). We focused on the L-type channel, Cav1.2, since CaM regulation of this channel has been studied extensively (reviewed in Halling et al., 2006). The IQ motif in Cav1.2 binds CaM, although sequences N-terminal to this sequence (pre-IQ), also appear to contribute to CaM binding (Erickson et al., 2003). We prepared two MBP fusion proteins: one that contained the IQ motif only and the other consisted of the pre-IQ and IQ sequence (pre-IQ/IQ). CaM binding to both fusion proteins was disrupted by PIP3, although the effect of this competition was much more pronounced using the pre-IQ/IQ fusion (Figure 7). In combination, these data indicate that regulation of CaM binding by PIs is common to multiple classes of cation channels.


Many TRP channels appear to be regulated by PIs, such as PIP2 or PIP3, although the mechanisms underlying PI-mediated regulation of TRP channels have not been described. Also unclear is whether or not PIP2/PIP3 act directly or indirectly on the channels. If TRP channels bind PIs, the question arises as to the mechanism through which PI/TRP interactions regulate channel activity. Multiple TRP channels have been reported to be positively regulated by PIP2 or PIP3 (Runnels et al., 2002; Liu and Liman, 2003; Tseng et al., 2004; Lee et al., 2005; Liu and Qin, 2005; Rohács et al., 2005; Nilius et al., 2006; Penna et al., 2006) and negatively regulated by Ca2+/CaM (Zhu, 2005), raising the possibility that the apparent opposite actions of these molecules are coordinated.

In the current work, we demonstrate that PIP3 binds directly to TRPC6 and interferes with CaM binding to the channel. Mutations in TRPC6 that increase or decrease the potency of the PI-mediated disruption of CaM binding (PDC) result in an enhancement or suppression of the TRPC6 current respectively. In support of these conclusions, we found that the C-terminal region of TRPC6 bound to PIs and potently disrupted CaM binding. Moreover, PIP3 was the PI, which was most effective in binding to TRPC6 and disrupting CaM binding. We found that PIP3 interfered with the CaM/TRPC6 interaction since the PI and CaM binding sites overlapped (PCaM domain). Nevertheless, it was possible to generate mutations that had differential impacts on PI or CaM binding (TRPC6R853Q and TRPC6R865Q respectively). Consequently, the potency through which PIP3 competed for CaM binding to TRPC6 was decreased nearly 10-fold in TRPC6R853Q and increased four-fold in TRPC6R865Q. Using these two TRPC6 derivatives, we found that the amplitude of the TRPC6-dependent current was decreased in TRPC6R853Q and increased in TRPC6R865Q. These effects were due to perturbation of CaM binding, rather than effects on surface expression as the mutations in TRPC6 had no impact on the expression or spatial distribution of the channels.

Despite the higher potency of PIP3 relative to PIP2 for reducing CaM binding, we cannot exclude that PIP2 is a physiologically relevant PI in vivo since it is significantly more abundant than PIP3 in most membranes. The levels of PIP2 change due to activity of phospholipase C. Increases in phospholipase C activity would decrease the PIP2 concentration and result in negative feedback regulation as a consequence of increased CaM mediated inhibition. Alternatively, regulation could be mediated by PIP3, if this PI is enriched in membrane domains in the vicinity of TRPC6 complexes. Interestingly, TRPC6 is activated in neutrophils through a pathway that results in stimulation of phosphatidylinositol 3-kinase (PI 3-kinase) (McMeekin et al., 2006). An elevation in PI 3-kinase activity would augment the TRPC6 current due to PIP3 mediated release of inhibitory Ca2+/CaM from the channel. An increase in Ca2+/CaM binding would result from an elevation of phospholipase C activity and TRPC6-mediated Ca2+ entry. Thus, PIP3 regulated control of CaM binding provides a mechanism for integration of channel regulation by PIP3 and Ca2+/CaM. Nevertheless, regulation of TRPC6 by CaM is complex as TRPC6 activity has been suggested to be increased by phosphorylation through CaM-dependent protein kinase II (Shi et al., 2004). TRPC6 may also associate with the Ca2+-CaM regulated phosphatase, calcineurin (Kim and Saffen, 2005).

We propose that the phenomenon of PIP3 mediated disruption of CaM binding is not specific to TRPC6, but is a common mode of regulation that applies to other TRP channels, and even to voltage-gated channels. Consistent with this proposal, TRPC1, TRPC7, TRPV1, KCNQ1 and Cav1.2 bound directly to PIP3 and such interactions interfered with CaM binding. Furthermore, as was the case for TRPC6, the PIP3 and CaM binding to these channels may occur through overlapping sequences C-terminal to the transmembrane segments. PI mediated regulation of CaM binding might extend to many additional TRP channels that have not yet been tested. Examples include TRPV5, which is positively regulated by PIP2, and TRPV2, whose activity is promoted by PI 3-kinase activity (Lee et al., 2005; Penna et al., 2006). The stimulation of TRPV2 by PI 3-kinase may occur through disruption of CaM binding since this effect is not due to increased translocation to the plasma membrane (Penna et al., 2006). Other candidate TRP channels that may be subject to PDC are TRPM4, TRPM5, TRPM7 and TRPM8, all of which are positively regulated by PIP2 (Runnels et al., 2002; Liu and Liman, 2003; Liu and Qin, 2005; Rohács et al., 2005; Zhang et al., 2005; Nilius et al., 2006; Penna et al., 2006). Moreover, PIP2 reduces desensitization of both TRPM4 and TRPM5 to Ca2+ (Liu and Liman, 2003; Zhang et al., 2005; Nilius et al., 2006). These data raise the possibility that TRPM4 and TRPM5 are desensitized through binding of Ca2+/CaM, and this effect is reversed by PIP2-mediated dissociation of the TRPM/CaM interaction.

The effect of PIP2 on TRPV1 activity appears to be opposite to that of many TRP channels, as PIP2 has been reported to be inhibitory, since it reduces the sensitivity to capsaicin and raises the temperature threshold for activation (Prescott and Julius, 2003). However, re-synthesis of PIP2 is required for recovery from desensitization (Liu et al., 2005). Since Ca2+/CaM is required for desensitization of TRPV1 (Numazaki et al., 2003), we suggest that PDC contributes to recovery from desensitization.

In addition to TRP channels, KCNQ1 and Cav1.2, many additional classes of ion channels and ion transporters appear to bind PIs and CaM (reviewed in Saimi and Kung, 2002; Suh and Hille, 2005; Halling et al., 2006). We suggest that regulation of ion fluxes through PIP2/PIP3-mediated regulation of CaM binding may extend to many additional ion channels and transporters that have not yet been tested.

The mode of regulation described in the current report may have relevance to understanding the bases of channelopathies resulting from mutations in PI and CaM binding sites. For example, Andersen’s and Bartter’s syndromes are associated with mutations that decrease PIP2 interactions with an inwardly rectifying K+ channel (Lopes et al., 2002). Other mutations that affect PIP2 or CaM binding to KCNQ1 underlie certain forms of the long QT syndrome and can result in sudden cardiac death (Park et al., 2005; Ghosh et al., 2006; Shamgar et al., 2006). PIP2 normally stabilizes the open state of KCNQ1. Whether mutations in the PCaM domain of TRPC6 leads to autosomal dominant segmental glomerulosclerosis remains to determined. Nevertheless, the findings in the current study indicate that the bases of channelopathies associated with disruption of PI binding may need to be reevaluated in terms of potential effects of these mutations on the stabilities of CaM with the effected channels.

Experimental Procedures

Purification of MBP fusions and mutagenesis

To generate MBP fusion proteins, the specified sequences were amplified by PCR and subcloned into pMAL-c2E (New England Biolabs). The fusion proteins were expressed in E.coli and purified using amylose resin according to the manufacturer’s instructions. The following MBP fusions were generated and purified: 1) MBP, 2) MBP-TRPC6-C (aa 728–931; human), 3) MBP-TRPC7-C (aa 673–862; mouse), 4) MBP-TRPC1-C (aa 612–759; human), 5) MBP-TRPC5-C (aa 625–975; mouse), 6) MBP-TRPV1-C (aa 684–838; rat), 7) MBP-KCNQ1-CBS1 (aa 345–400; human), 8) MBP-KCNQ1-CBS2 (aa 504–565; human), 9) MBP-Cav1.2-preIQ/IQ (aa 1558–1674; rabbit), and 10) MBP-Cav1.2-IQ (aa 1635–1674; rabbit). Point mutations were introduced into the TRPC6 cDNA by performing PCR reactions with mismatched primers.

Inositol polyphosphate binding assay

[3H]IP6 binding to the MBP fusions was determined by performing pull-down assays. Briefly, 2 μg of purified MBP fusions were incubated with 0.01μCi [3H]IP6 (NEN) for 40 min at 4 °C in 200 μl of IP6 binding buffer [25 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/ml bovine γ-globulin, 0.25% Triton X-100 and protease inhibitor (Sigma P2714)]. The MBP fusions were bound to amylose resin (New England Biolabs), washed twice in 1 ml binding buffer and the pellets were resuspended in 200 μl of 1% SDS to dissociate the MBP fusions from the resin. Radioactivity was measured using a scintillation counter. To test for competition of [3H]IP6 by unlabeled IP6 and PIs, the indicated concentrations of IP6 and short chain (diC8) PIs (Echelon Bioscience) were combined with 0.01 μCi of [3H]IP6 during incubation. Centrifugations were performed at 2000 rpm for 3 min.

Binding of fusion proteins to phospholipids immobilized on nitrocellulose filters

Binding of recombinant proteins to phospholipids immobilized on nitrocellulose filters were performed essentially as described (Lee et al., 2003). Briefly, PIP Strips® (Echelon Bioscience; P-6001) were blocked with 3% fatty acid-free BSA (Sigma; A-6003) in TBST [50mM Tris (pH 7.5), 150 mM NaCl, 0.25% Triton X-100 and protease inhibitor] and incubated with purified MBP fusions (0.5 μg/ml) for 2 hr at 4ºC in TBST. The membranes were washed 3x in TBST, incubated with rabbit anti-MBP antibodies (1:5000; New England Biolabs) in TBST for 1 hr at RT, washed 3x and incubated with horseradish peroxidase-coupled anti-rabbit antibodies (1:10000; GE healthcare) for 1 hr at RT. After washing 3x, the MBP fusions which were bound to the filters were determined using enhanced chemiluminescence (ECL; GE healthcare).

Calmodulin binding assays

CaM binding to the MBP fusion proteins was determined by performing pull-down assays using CaM-Sepharose 4B (GE Healthcare). The resin was equilibrated in binding buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 0.25% Triton X-100, protease inhibitor] and 60 μl of CaM-Sepharose slurry was incubated for 1 hr at 4 ºC with 200 ng of the MBP fusion proteins in 300 μl of binding buffer. After washing 4x in 1 ml of binding buffer, the bound fusion proteins were eluted by addition of 100 μl of 2x SDS sample buffer. 10 μl/sample were fractionated by SDS-PAGE and the MBP fusion proteins were detected by Western blotting using anti-MBP antibodies. To test for competition of CaM binding, various concentrations of IP6 (Sigma) and short chain (diC8) PIs (Echelon Bioscience) were added during the incubations with the MBP fusion proteins. Quantification of the Western blot signals were performed by scanning the X-ray films and assessing the intensities of the bands using ImageJ software (NIH, USA)

Cell culture and transfection

HEK293T cells were maintained in DMEM with 10% fetal bovine serum and penicillin/streptomycin. Transfections were performed on 35 mm culture dishes using the FuGENE6 transfection reagent (Boehringer/Mannheim) and 2 μg of guinea pig H1 histamine receptor cDNA and 2 μg of pcDNA3-human TRPC6 (wild-type or mutants)-YFP. After incubating overnight, the cells were split onto glass coverslips and used within 18–30 hr. In some experiments, the TRPC6 constructs were co-transfected with either 2 μg of pcDNA-AKT-PH-myc (wild-type or mutant) or 3 μg of pcDNA3-CaM-YFP. Co-transfection of 3 μg of the empty pcDNA3 vector with TRPC6 (wild-type or mutants)-YFP induced similar currents as in cells transfected with the TRPC6 vectors alone.


The patch-clamp pipets were prepared from borosilicate glass capillary tubing (Sutter instrument company) and had a resistance of 2.5–4.5 M after they were filled with the solution. The data were recorded using an Axopatch 200B amplifier (Axon instruments), digitized with an ITC18 AD/DA converter (Instrutech), and stored with the Pulse/Pulsefit software (HEKA Elektronik). The data were filtered at 1 kHz with an eight-pole Bessel filter (Frequency devices) and sampled at 3.3 kHz. The holding potential was -60 mV. The series resistances ranged between 4 and 10 M and were compensated >95%. The bath solution contained 140 mM NaCl, 5 mM CsCl2, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose. The pipet solution contained 120 mM CsCl2, 10 mM NaCl, 1 mM MgCl2 and 10 mM HEPES [pH 7.4]. To buffer intracellular Ca2+, 20 mM BAPTA was included in the recording pipette solution. 5–7 sec after breaking a seal, the cells were stimulated with 100 μM histamine, or left unstimulated.

Surface biotinylation

HEK293T cells were transfected with the H1 receptor cDNA and one of the following DNAs inserted into pcDNA3.1: hTRPC6-YFP or derivatives, mouse CD8-YFP or Venus. The cells were washed 2x in HBS (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 10 mM HEPES at pH 7.4) and stimulated with a final concentration of 100 μM histamine as indicated. The reactions were stopped after 20 sec by aspiration of the saline and by immediate cooling on an ice/water bath. The cells were washed 3x with ice-cold PBS (pH 8.0) and labeled with biotin [3 mg NHS-LC-biotin (Pierce) in 4 ml of PBS each] at 4 °C for 1 hr. The reactions were quenched with 100 mM glycine in PBS, the cells were washed 2x in PBS, and lysed with 0.5 ml RIPA buffer (150 mM NaCl, 50 mM Tris/HCl at pH 8.0, 1 % NP-40, 0.5 % sodium deoxycholate and 0.1 % SDS) supplemented with the Complete protease inhibitor cocktail (Roche). The lysates were centrifuged for 10 min at 8000 g to remove debris and 2 hrs at 50,000 g to remove incompletely solubilized microsomes. 20 μl of the lysates were retained for Western blots; the remainders of the lysates were shaken overnight at 4 °C with 25 μl of NeutrAvidin agarose beads. The precipitates were pelleted, washed 3x in RIPA buffer, denaturated in Laemmli buffer and fractionated by SDS-PAGE along with the lysate samples. The Western blots were probed with rabbit anti-GFP antibodies (Invitrogen, 1:2000) and with goat anti-rabbit IgG antibodies. The bands were detected using a pico chemiluminescence substrate kit (Pierce). Mouse CD8-YFP was used as an internal positive control for surface labeling and Venus as a negative control. Both controls were detected with the same antibodies used to detect hTRPC6-YFP.

Detection of AKT-PH-GFP

AKT-PH-GFP (Botelho et al., 2000) and AKT-PHR25C-GFP (Franke et al., 1997) were transfected together with the H1 receptor in HEK293T cells. 24 hours later the cells were analysed with a Zeiss LSM510 meta confocal laser scanning microscope using the 488 nm excitation line of the argon laser and the pinhole setting to achieve sections thinner than 1 μm.

Supplementary Material



We thank Drs. E. Marbán for the KCNQ1 cDNA, D. Yue for the Cav1.2 and CaM-YFP cDNA and S. Kim for the AKT-PH cDNA. We also thank Drs. M. Caterina and M. Köttgen for helpful comments on the manuscript. This research was supported by a grant from the NEI (EY10852) to CM.


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