1. Integration of Phosphoinositide- and Calmodulin-Mediated Regulation of TRPC6 
Young Kwon, Thomas Hofmann, Craig Montell 
Molecular Cell 
Volume 25, Issue 4, Pages (February 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. 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 anti-rabbit antibodies. Signals were detected by ECL.
(B) Solution binding of [3H]IP6 to purified MBP-TRPC6-C. Mean CPMs (±SEMs) were based on three or more independent sets of experiments (MBP = 75.3 ± 4.4; MBP-TRPC6-C = ± 17.7). The asterisk indicates a statistically significant difference from wild-type using the unpaired Student's t test, value < 0.05.
(C) PIP2 or PIP3 reduced binding of [3H]IP6 to TRPC6-C in a concentration-dependent manner. Binding of [3H]IP6 to MBP-TRPC6-C in the presence of indicated concentrations of PIP2 or PIP3.
(D) IC50 values indicating efficacy by which PIs competed for [3H]IP6 binding to TRPC6 (n ≥ 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
PI- and CaM-Binding Domains Overlapped in the TRPC6 C Terminus
(A) Schematic of C-terminal regions of TRPC6 (C6-C) fused to MBP. The TRP domain and CaM-binding domain (CBD) are indicated. The residues in TRPC6 included in the fusions are indicated. The sixth transmembrane domain is predicted to end at residue 727.
(B) Relative IP6 binding to the MBP-TRPC6-C fusion proteins. The bindings were based on determination of the CPMs using [3H]IP6 (±SEMs; n ≥ 3): C6-C, ± 23.8; fragment 1, ± 13; fragment 2, ± 5.9; fragment 3, 36.9 ± 8.6; fragment 4, 30.7 ± 18.6; fragment 5, 125.3 ± 12.4; and fragment 6, 236 ± 7.4.
(C) PCaM domain. Indicated in bold are the positively charged amino acids mutated.
(D) Relative IP6 binding to wild-type and mutant MBP-TRPC6-C fusion proteins. The relative bindings were based on determination of the CPMs using [3H]IP6 (±SEMs; n ≥ 3): wild-type, ± 2.6; R853Q, 36.7 ± 4.4; K860Q, 85.9 ± 2.8; R861Q, 76.5 ± 3.2; R865Q, 97.3 ± 13.7; R853Q/K860Q, 22.1 ± 3.6; R853Q/R861Q, 30.3 ± 3.1; K860Q/R861Q, 62.3 ± 5.3; and R853Q/K860Q/R861Q, 24.9 ± 4.1. Asterisks indicate statistically significant differences from wild-type using the unpaired Student's t test, value < 0.05.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. 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 was based on the data in Figures 3B and 3C. The [3H]IP6 binding was based on Figure 2D. The data summarizing the PIP3-mediated disruption of CaM binding to MBP-TRPC6-C (PDC50) were based on Figures 3D and 3E. nd, not done.
(B) CaM-Sepharose pull-down assay. The bound wild-type and mutant MBP-TRPC6-C proteins were detected by western blotting using anti-MBP antibodies.
(C) Relative binding of the wild-type and mutant MBP-TRPC6-C proteins to CaM-Sepharose. The data were based on western blots from the following (n ≥ 3; percent ± SEM): wild-type, ± 0.7; R853Q, 69.2 ± 3.1; R865Q, 64.9 ± 1.6; R853Q/K860Q/R861Q, 3.7 ± 0.5. Asterisks indicate statistically significant differences from wild-type using the unpaired Student's t test, value < 0.05.
(D) Interference of CaM binding to wild-type and mutant TRPC6-C (R853Q and R865Q) by PIs/IP6. Fusion proteins were bound to CaM-Sepharose in the presence of the indicated concentrations of IP6, PI, PIP2, and PIP3, eluted and detected by western blotting. The PDC50 values (μM) were as follows: (wild-type) IP6 > 20, PI > 20, PIP2 = 8.2, and PIP3 = 1.2; (R853Q) PIP2 = 16.3 and PIP3 = 11.0; and (R865Q) PIP2 = 1.2 and PIP3 = 0.3.
(E) Interference of CaM binding to MBP-TRPC6-C by various concentrations of PIs/IP6 (n ≥ 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. 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. Gray bars (middle bars in each set) indicate the average initial current amplitudes in HEK293T cells expressing TRPC6 derivatives (nA ± SEMs): wild-type TRPC6 (wild-type), 0.91 ± 0.16, n = 13; TRPC6R853Q (R853Q), 0.31 ± 0.07, n = 12; TRPC6R865Q (R865Q), 1.92 ± 0.44, n = 17; and TRPC63Q (R853Q/K860Q/R865Q), 1.86 ± 0.32, n = 14. Red asterisks indicate statistically significant differences from wild-type using the unpaired Student's t test (value < 0.05). Black bars indicate the average current amplitudes resulting from TRPC6 activation after buffering intracellular Ca2+, using 20 mM BAPTA in the recording pipettes (nA ± SEMs): wild-type, 2.02 ± 0.18, n = 6; R853Q,1.83 ± 0.35, n = 7; R865Q, 2.33 ± 0.49, n = 7; and R853Q/K860Q/R865Q, 2.42 ± 0.58, n = 5. White bars indicate the average current amplitudes resulting from TRPC6 activation with CaM overexpression (nA ± SEMs): wild-type, 0.21 ± 0.03, n = 7; R853Q, 0.16 ± 0.03, n = 7; R865Q, 1.27 ± 0.34, n = 9; and R853Q/K860Q/R865Q, 1.48 ± 0.28, n = 6. Black asterisks and brackets indicate statistically significant differences between two measurements within a group using the unpaired Student's t test (value < 0.05).
(B) Representative IV curves obtained at the peak currents in cells expressing TRPC6 channels in the absence of Ca2+ buffering or CaM overexpression.
(C–F) Representative current traces resulting from stimulation of TRPC6 channels in the absence of Ca2+ buffering or CaM overexpression. The channels were coexpressed in HEK293T cells with the H1 histamine receptor and were activated by applying 100 μM histamine. (C) Wild-type TRPC6. (D) TRPC6R853Q (R853Q). (E) TRPC6R865Q (R865Q). (F) TRPC63Q (R853Q/K860Q/R861Q).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. 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 were transfected into HEK293T cells, and the GFP signals in live cells were detected using confocal microscopy. The mutant AKT-PHR25C-GFP, which does not bind PIP3, was detected at the cytosol.
(B) Average current amplitudes resulting from histamine stimulation of cells expressing TRPC6 channels, along with either AKT-PH-myc (black) or AKT-PHR25C-myc (gray) overexpression (nA ± SEMs): (1) wild-type TRPC6-AKT-PHR25C, 1.12 ± 0.19, n = 6; AKT-PH, 0.47 ± 0.14, n = 6; (2) TRPC6R853Q (R853Q)-AKT-PHR25C, 0.22 ± 0.05, n = 6; AKT-PH, 0.25 ± 0.05, n = 5; (3) TRPC6R865Q (R865Q)-AKT-PHR25C, 1.86 ± 0.35, n = 6; AKT-PH, 1.19 ± 0.32, n = 5; and (4) TRPC63Q (R853Q/K860Q/R865Q)-AKT-PHR25C, 1.89 ± 0.33, n = 6; AKT-PH, 2.01 ± 0.38, n = 5. The asterisk indicates a statistically significant difference (unpaired Student's t test, <0.05).
(C–F) Representative current traces resulting from histamine stimulation of cells expressing TRPC6 channels with either AKT-PH (black traces) or AKT-PHR25C (gray traces). The horizontal bars indicate application of 100 μM histamine. (C) Wild-type TRPC6. (D) TRPC6R853Q (R853Q). (E) TRPC6R865Q (R865Q). (F) TRPC63Q (R853Q/K860Q/R865Q).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
PI/IP 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 residues 728–931), TRPC7 (C7-C; mouse residues 673–862), and TRPV1 (V1-C; rat residues 684–838). CPMs (±SEMs; n ≥ 3): MBP only, 29.3 ± 3.8; TRPC1-C, ± 74.7; TRPC5-C, ± 27.0; TRPC6-C, ± 19.1; TRPC7-C, ± 38.0; and TRPV1-C, ± The asterisks indicate statistically significant differences in [3H]IP6 binding between MBP alone and the fusion proteins, using the unpaired Student's t test (value < 0.05).
(B) Binding of MBP-TRPC1-C, MBP-TRPC5-C, and MBP-TRPC7-C to PIP Strips. The fusion proteins were detected by ECL after probing with anti-MBP antibodies.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. 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 MBP-TRP fusions were used as described in Figure 6. The KCNQ1 and Cav1.2 sequences included in the MBP fusions were as follows: KCNQ1-CBS1 (human residues 345–400), KCNQ1-CBS2 (human residues 504–565), Cav1.2-pre-IQ/IQ (rabbit residues 1558–1674), and Cav1.2-IQ (rabbit residues 1635–1674). The bound and eluted proteins were detected on western blots using anti-MBP antibodies.
(B) Relative binding of the indicated MBP fusions to CaM-Sepharose in the presence of IP6/PIs. The TRPC6-C data were based on the experiments in Figures 3D and 3E and are included for comparison. The following are the mean values (percent ± SEMs; n ≥ 3) obtained in the absence of any added IP6/PIs or the presence of either 20 μM IP6, 20 μM PIP2, or 20 μM PIP3: (MBP-TRPC6-C) ± 2.5, 73.8 ± 8.9, 89.5 ± 0.6, 29.6 ± 7.6, and 10.1 ± 4.5; (MBP-TRPC1-C) ± 11.3, 94.4 ± 6.2, 94.3 ± 1.6, 46.9 ± 6.8, and 38.4 ± 12.0; (MBP-TRPC5-C) ± 11.3, 90.6 ± 6.2, ± 1.6, 99.2 ± 6.8, and 95.6 ± 12.0; (MBP-TRPC7-C) ± 1.9, 97.7 ± 0.9, 95.0 ± 2.5, 68.0 ± 1.9, and 53.7 ± 4.6; (MBP-TRPV1-C) ± 6.3, 67.7 ± 9.1, 93.3 ± 4.7, 34.3 ± 5.5, and 18.0 ± 2.7; (MBP-KCNQ1-CBS1) ± 5.8, 99.6 ± 10.8, ± 7.9, 61.4 ± 2.4, and 31.8 ± 6.7; (MBP-KCNQ1-CBS2) ± 4.2, ± 6.6, 95.5 ± 16.8, ± 6.7, and 53.5 ± 4.5; (MBP-Cav1.2-pre-IQ/IQ) ± 1.1, ± 4.3, 94.6 ± 2.3, 54.6 ± 5.6, and 7.8 ± 0.9; and (MBP-Cav1.2-IQ) ± 2.5, 94.1 ± 6.7, 96.9 ± 5.2, 72.3 ± 5.0, and 40.1 ± 1.6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions