Activation of TRPP2 through mDia1-dependent voltage gating
2008; Springer Nature; Volume: 27; Issue: 9 Linguagem: Inglês
10.1038/emboj.2008.70
ISSN1460-2075
AutoresChang-Xi Bai, Sehyun Kim, Wei Ping Li, Andrew J. Streets, Albert Ong, Leonidas Tsiokas,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoArticle3 April 2008free access Activation of TRPP2 through mDia1-dependent voltage gating Chang-Xi Bai Chang-Xi Bai Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Sehyun Kim Sehyun Kim Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Wei-Ping Li Wei-Ping Li Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USAPresent address: Department of Pharmacology, Anhui Medical University, Hefei, Anhui Province 230032, People's Republic of China Search for more papers by this author Andrew J Streets Andrew J Streets Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK Search for more papers by this author Albert C M Ong Albert C M Ong Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK Search for more papers by this author Leonidas Tsiokas Corresponding Author Leonidas Tsiokas Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Chang-Xi Bai Chang-Xi Bai Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Sehyun Kim Sehyun Kim Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Wei-Ping Li Wei-Ping Li Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USAPresent address: Department of Pharmacology, Anhui Medical University, Hefei, Anhui Province 230032, People's Republic of China Search for more papers by this author Andrew J Streets Andrew J Streets Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK Search for more papers by this author Albert C M Ong Albert C M Ong Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK Search for more papers by this author Leonidas Tsiokas Corresponding Author Leonidas Tsiokas Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Search for more papers by this author Author Information Chang-Xi Bai1, Sehyun Kim1, Wei-Ping Li1, Andrew J Streets2, Albert C M Ong2 and Leonidas Tsiokas 1 1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA 2Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK *Corresponding author. Department of Cell Biology, University of Oklahoma Health Sciences Center, 975 NE 10th str, BRC1/262, Oklahoma City, OK 73104, USA. Tel.: +01 405 271 8001 ext 46211; Fax: +01 405 271 3758; E-mail: [email protected] The EMBO Journal (2008)27:1345-1356https://doi.org/10.1038/emboj.2008.70 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The TRPP2 cation channel is directly responsible for ∼15% of all cases of autosomal dominant polycystic kidney disease. However, the mechanisms underlying fundamental properties of TRPP2 regulation, such as channel gating and activation, are unknown. We have shown that TRPP2 was activated by EGF and physically interacted with the mammalian diaphanous-related formin 1 (mDia1), a downstream effector of RhoA. Now, we show that mDia1 regulates TRPP2 by specifically blocking its activity at negative but not positive potentials. The voltage-dependent unblock of TRPP2 by mDia1 at positive potentials is mediated through RhoA-induced molecular switching of mDia1 from its autoinhibited state at negative potentials to its activated state at positive potentials. Under physiological resting potentials, EGF activates TRPP2 by releasing the mDia1-dependent block through the activation of RhoA. Our data reveal a new role of mDia1 in the regulation of ion channels and suggest a molecular basis for the voltage-dependent gating of TRP channels. Introduction TRPP2 (polycystin-2 or PKD2) is the protein product of the pkd2 gene originally identified as one of the genes responsible for autosomal dominant polycystic kidney disease (Mochizuki et al, 1996). Structurally, it belongs to the TRP superfamily of channel proteins (Nilius et al, 2007; Venkatachalam and Montell, 2007) and possesses channel activity (Hanaoka et al, 2000; Gonzalez-Perrett et al, 2001; Vassilev et al, 2001; Koulen et al, 2002; Luo et al, 2003; Delmas et al, 2004; Ma et al, 2005), which mediates all of its biological functions (Cantiello, 2004; Tsiokas et al, 2007). However, some of the very basic biophysical properties of TRPP2, such as gating and activation, remain completely unknown. Recent study on a subset of TRP channels activated mainly by temperature has shown that voltage-dependent gating underlies their activation mechanism (Voets et al, 2004; Nilius et al, 2005). Current data on the voltage-dependent gating of TRPP2 are quite controversial depending on the cell or cell-free system used. In vitro-reconstituted TRPP2 derived from various sources such as endoplasmic reticulum endomembranes (Koulen et al, 2002), Sf9 insect plasma membranes, apical membranes of syncytiotrophoblasts (Gonzalez-Perrett et al, 2001, 2002) or in vitro-translated TRPP2 (Gonzalez-Perrett et al, 2001) showed strong voltage dependence with high activity at negative potentials and almost no activity at positive potentials. In sharp contrast, native TRPP2 displayed voltage dependence with higher activity at positive potentials (Luo et al, 2003; Pelucchi et al, 2006; Kim et al, 2008) and overexpressed TRPP2 alone or in association with PKD1 did not show voltage dependence (Hanaoka et al, 2000; Delmas et al, 2004; Ma et al, 2005). It has been proposed that voltage-dependent gating of TRPP2 could be an intrinsic property of the channel, but it should be greatly modulated by protein–protein interactions under physiological conditions (Gonzalez-Perrett et al, 2001; Cantiello, 2004). TRPP2 activation was shown to occur in response to EGFR activation in the kidney epithelial cell line, LLC-PK1 (Ma et al, 2005). The physiological relevance of EGF-induced activation of TRPP2 in LLC-PK1 cells was supported by whole animal studies whereby homozygous deletion of egfr resulted in cystic dilatation of collecting ducts (Threadgill et al, 1995), an area that was also predominantly affected by pkd2 mutations (Wu et al, 2000). Mechanistically, TRPP2 overexpression increased EGF-induced conductance in LLC-PK1 kidney epithelial cells, whereas knock down of endogenous TRPP2 by RNA interference (RNAi) or expression of the pathogenic, missense variant, TRPP2-D511V, blunted the EGF-induced response (Ma et al, 2005). Pharmacological experiments indicated that the EGF-induced activation of TRPP2 occurred independently of store depletion but required the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) (Ma et al, 2005). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP2) suppressed the TRPP2-mediated effect on EGF-induced conductance, whereas pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP3) did not have any effect on this conductance (Ma et al, 2005). Overexpression of type Iα phosphatidylinositol-4-phosphate 5-kinase (PIP(5)Kα), which catalyses the formation of PIP2, suppressed EGF-induced TRPP2 currents (Ma et al, 2005). Overall, TRPP2 functioned downstream of EGFR activation in LLC-PK1 cells. In a yeast two-hybrid screen using the C-terminal tail of TRPP2, we identified mammalian diaphanous-related formin 1 (mDia1) as an interacting partner (Rundle et al, 2004). mDia1 functions downstream of RhoA in signal transduction, cytoskeletal organization, and cell cycle regulation (Wallar and Alberts, 2003; Gundersen et al, 2004; Higgs, 2005; Narumiya and Yasuda, 2006). Inactive or autoinhibited mDia1 exists in a 'closed' conformational state, whereby the diaphanous autoregulatory domain (DAD) at its C terminus loops around to bind the diaphanous inhibitory domain (DID) at its N terminus (Watanabe et al, 1999; Lammers et al, 2005; Otomo et al, 2005; Rose et al, 2005). Activated, GTP-bound Rho proteins (RhoA-C) bind to a G (for GTPase binding) domain, which is in close proximity to DID and relieves inhibition by DAD resulting in activation of mDia1. Activated mDia1 assumes an 'open' conformation, exposing formin homology 1 and 2 (FH1 and FH2) domains for interaction with several downstream effector molecules. In the present study, we show that mDia1 functioned as an intracellular, voltage-dependent regulator of TRPP2. At resting potentials, autoinhibited mDia1 bound to and blocked TRPP2, whereas at positive potentials activated mDia1 released the block on TRPP2 leading to channel activation. EGF or membrane depolarization activated TRPP2 through the sequential activation of RhoA and mDia1. The voltage-dependent block of TRPP2 by mDia1 served two physiologically relevant roles, to underlie the activation mechanism of TRPP2 by EGF and to set the resting membrane potential of kidney epithelial cells to its resting value by preventing constitutive activation of TRPP2. Results Colocalization of native TRPP2 and mDia1 in the plasma membrane of kidney epithelial cells Although TRPP2 has been shown to be present in the plasma membrane of a variety of cell types, including MDCK (Scheffers et al, 2002), mIMCD3 (Luo et al, 2003), HEK293 (Pelucchi et al, 2006), and human syncytiotrophoblasts (Gonzalez-Perrett et al, 2001), its presence in the plasma membrane of LLC-PK1 cells has been questionable (Koulen et al, 2002; Luo et al, 2003). Immunofluorescence staining of endogenous TRPP2 in LLC-PK1 cells using a commercially available antibody (G20; Santa Cruz Biotechnology Inc.) revealed consistent staining in the plasma membrane (Figure 1A and D), as was previously shown for MDCK and mIMCD3 cells (Li et al, 2005a). G20 detected native and transfected TRPP2 (Figure 1C). Plasma membrane staining was specific to TRPP2, as it was eliminated in cells treated with the glycogen synthase kinase 3 (GSK3) inhibitor (Figure 1B), SB 415286, which was shown to inhibit the GSK3-dependent N-terminal phosphorylation of TRPP2 necessary for its targeting to the plasma membrane in MDCK cells (Streets et al, 2006). To determine whether mDia1 colocalized with TRPP2 at the plasma membrane, native LLC-PK1 cells were double stained with G20 (α-TRPP2) and a mouse monoclonal antibody against mDia1. Figure 1D–F shows that both proteins colocalized at the plasma membrane. Transfected TRPP2 was also detected at the plasma membrane and its expression there was not altered by transfected mDia1 (Supplementary Figure S1). Altogether, these data led us to conclude that endogenous or transfected TRPP2 was expressed in a significant amount at the plasma membrane of LLC-PK1 cells where it colocalized with mDia1. mDia1 did not affect TRPP2 trafficking to and/or from the plasma membrane. Figure 1.Colocalization of mDia1 and TRPP2 at the plasma membrane of LLC-PK1 cells. Expression of endogenous TRPP2 in untreated LLC-PK1 cells (A) or cells treated with the GSK3 inhibitor, SB 415286 (40 μM) for 16 h (B). (C) Detection of endogenous (lane 1) or transfected TRPP2 (lane 2) in lysates (75 μg) of LLC-PK1 cells by α-TRPP2 (1:300, G20; Santa Cruz Biotechnology Inc.). (D–F) Cell surface colocalization of native TRPP2 and mDia1 in LLC-PK1 cells. TRPP2 was stained with G20 (1:50) (D) and mDia1 with a mouse monoclonal (1:500) (E). Overlay is shown in (F). Download figure Download PowerPoint Voltage-dependent regulation of TRPP2 by mDia1 in native LLC-PK1 cells Next, we measured whole cell background currents in native LLC-PK1 cells. Cells were held at −60 mV and bathed in normal tyrode solution. Under these conditions, an outwardly rectifying current was recorded in native LLC-PK1 cells (Figure 2A and B). To determine whether native TRPP2 contributed to this current, we employed two independent loss-of-function experiments. First, LLC-PK1 cells were transiently transfected with a single-point mutant form of TRPP2 (TRPP2-D511V). This mutant lacks channel activity (Koulen et al, 2002; Ma et al, 2005; Li et al, 2005b) and behaves as a dominant-negative interfering allele (Ma et al, 2005) by binding up wild-type TRPP2 (Supplementary Figure S2A, lane 2). Second, LLC-PK1 cells were dialysed through pipette infusion with a rabbit TRPP2-specific affinity-purified antibody (α-TRPP2) which has been shown previously to neutralize TRPP2 activity (Ma et al, 2005). Transfection of TRPP2-D511V (Figure 2C and D) or pipette infusion of α-TRPP2, but not control IgG (Supplementary Figure S2B–E) reduced both inward and outward background whole cell currents. However, the effect was more pronounced at positive potentials (Supplementary Figure S2D and E), indicating that native TRPP2 contributed to an outwardly rectifying current. Next, we tested the role of endogenous mDia1 on native currents and its functional interaction with TRPP2. We designed an shRNAi construct to target porcine mDia1 (shRNAimDia1). The silencing efficiency of such a construct is shown in Supplementary Figure S2F. Knocking down mDia1 increased mainly inward currents (Figure 2E and F), whereas double transfection of TRPP2-D511V and shRNAImDia1 suppressed both inward and outward currents to levels seen with TRPP2-D511V alone (Figure 2G and H). These data showed that endogenous mDia1 blocked TRPP2-mediated inward, but not outward currents. Therefore, native TRPP2 alone or in association with other channel subunits was under voltage-dependent regulation by mDia1 in LLC-PK1 cells. Figure 2.Voltage-dependent block of endogenous TRPP2 by mDia1 in LLC-PK1 cells. (A–H) Step currents and pooled current–voltage (I–V) curves in normal tyrode extracellular solution in native LLC-PK1 (untransfected, n=9) (A, B), TRPP2-D511V-transfected LLC-PK1 cells (TRPP2-D511V, n=8) (C, D), shRNAimDia1-transfected cells (shRNAimDia1, n=8) (E, F), or TRPP2-D511V plus shRNAimDia1-doubly transfected cells (TRPP2-D511V plus shRNAimDia1, n=7) (G, H). Pooled I–V curve derived from native LLC-PK1 cells (B) is shown in black for comparison (D, F, and H). *P 10-fold) overexpression of TRPP2 in LLC-PK1 cells either by stable expression (LLC-PK1TRPP2 cells) (Ma et al, 2005) or transient transfection from a strong mammalian promoter (cytomegalovirus promoter, pCDNA3-based vectors, data not shown) resulted in the formation of a constitutively active channel with no obvious signs of any type of voltage dependence (Figure 3A and B). In addition, overall increase in whole cell outward currents was ∼50% (from 18.9±1.4 pA/pF in LLC-PK1 cells (n=9) to 28.6±0.6 pA/pF in LLC-PK1TRPP2 cells (n=9) at −100 mV) (Figure 3B), despite massive overexpression. To explain the lack of residual outward rectification and modest amplitude increase, we reasoned that TRPP2 could not form a functional channel by itself but it had to interact with additional subunits such as PKD1 (Tsiokas et al, 1997), TRPC1 (Tsiokas et al, 1999; Bai et al, 2008), TRPC4 (through an indirect interaction with TRPC1), and/or TRPV4 (Kottgen and Walz, 2005). Therefore, massive overexpression of just TRPP2 could alter the stoichiometry of native complexes and most importantly, strip mDia1 from these complexes. Monomeric TRPP2 bound to mDia1 should be non-functional, whereas TRPP2 associated with endogenous channel subunits should form functional complexes passing linear currents, as they would have lost regulation by mDia1. To provide evidence for this model, we expressed TRPP2 from the much weaker rat β-actin promoter (pJ6Ω expression plasmid, ATCC). Supplementary Figure S3A confirms that rat β-actin promoter was utilized less efficiently than the CMV promoter in LLC-PK1 cells, when the two promoters were compared side by side. Transfection of pJ6Ω-TRPP2 resulted in an amplification of native, outwardly rectifying currents (Supplementary Figure S3B–E), supporting the existence of a heteromultimeric channel complex of TRPP2 (Supplementary Figure S3F). We, therefore, proceeded with the LLC-PK1TRPP2 cells as an overexpression system, because it would allow us to add individual components (i.e. mDia1, RhoA constructs) and test whether linear TRPP2-mediated currents could be converted to outwardly rectifying currents. Figure 3.Voltage-dependent block of TRPP2 by activated mDia1. (A–J) Step currents and pooled I–V curve in normal tyrode extracellular solution in LLC-PK1TRPP2 cells (TRPP2, n=9) (A, B), LLC-PK1TRPP2 cells transiently transfected with mDia1 (TRPP2+mDia1, n=8) (C, D), transiently co-transfected with mDia1 plus RhoA(N19) (TRPP2+mDia1+RhoA(N19), n=8) (E, F), mDia1 plus RhoA(V14) (TRPP2+mDia1+ RhoA(V14), n=8) (G, H), or RhoA(V14) without mDia1 (TRPP2+RhoA(V14), n=7) (I, J). Pooled I–V curve derived from untransfected LLC-PK1 cells (Figure 2B) is shown in open circles for comparison (B). Pooled I–V curve derived from LLC-PK1TRPP2 cells (B) is shown in black for comparison (D, F, H, and J). *P<0.05. Parentheses indicate a range of membrane potentials at which there was a significance difference in current density between the two groups of cells. (K–R) Tail (Itail) and steady-state currents (Iss) in mDia1-untransfected (TRPP2, Cm=22.7±0.4 pF, n=8) (K, L), mDia1- (TRPP2+mDia1, Cm=22.8±0.4 pF, n=7) (M, N), mDia1 plus RhoA(V14)-transfected (TRPP2+mDia1+RhoA(V14), Cm=23.2±0.4 pF, n=7) (O, P), or RhoA(V14)- (TRPP2+RhoA(V14), Cm=22.9±0.5 pF, n=7) transfected LLC-PK1TRPP2 cells (Q, R). Tail-current protocol used to demonstrate the voltage-dependent gating of TRPP2 is shown in inset. *P<0.05. Parentheses indicate a range of membrane potentials at which there was a significance difference between tail and steady-state currents within the same group. Download figure Download PowerPoint Transient transfection of wild-type mDia1 (Figure 3C and D) into LLC-PK1TRPP2 cells suppressed both inward and outward currents, indicating that overexpression of the autoinhibited form of mDia1 was not sufficient to confer voltage sensitivity to TRPP2. Next, we examined whether activation of mDia1 could affect TRPP2 activity. Activation of mDia1 was achieved by co-transfection with constitutively active RhoA(V14), whereas dominant-negative RhoA(N19) was used as a negative control. Figure 3E and F shows that RhoA(N19) did not have an effect on mDia1-mediated block of TRPP2, whereas RhoA(V14) resulted in suppression of inward but not outward currents in LLC-PK1TRPP2 cells (Figure 3G and H), recapitulating native currents. Overexpression of RhoA(V14) in the absence of transfected mDia1 did not alter TRPP2-mediated currents (Figure 3I and J) confirming the role of mDia1 in TRPP2 regulation and also, the existence of a heteromultimeric complex of TRPP2 and other channel subunits (Supplementary Figure S3F). If TRPP2 associated with other channel subunits had retained some of native mDia1, massive expression of Rho(V14) should have resulted in some outward rectification, which was not the case. Confirmation that transfected mDia1 and RhoA(V14) acted through TRPP2 was obtained by the suppression of whole cell currents by cell dialysis of triple-transfected cells with α-TRPP2 (data not shown in normal tyrode solution, but shown in symmetrical K+; Supplementary Figure S5E and F). Slight current activation (or relaxation) at depolarizing potentials was only noted in cells co-transfected with mDia1 and RhoA(V14) (Figure 3G), but not with any of the cells transfected with TRPP2 alone (Figure 3A), TRPP2/mDia1 (Figure 3C), TRPP2/mDia1/RhoA(N19) (Figure 3E), or TRPP2/RhoA(V14) (Figure 3I). To test whether the activated mDia1-induced outward rectification (Figure 3G and H) was due to voltage-dependent gating of TRPP2, we employed a tail current protocol (Voets et al, 2004). Because, mDia1 was originally identified as an interacting partner of TRPP2 (Rundle et al, 2004), we hypothesized that activated mDia1 bound to and blocked TRPP2 activity at negative potentials, whereas it dissociated or 'swung away' from TRPP2 at positive potentials. Therefore, dissociation of activated mDia1 from TRPP2 should be manifested as time-dependent current relaxation at positive potentials, whereas protein re-association should be manifested as time-dependent current de-activation at hyperpolarizing potentials following a strong depolarizing pulse at 120 mV for 200 ms. Figure 3K–R shows that whole cell currents displayed current relaxation and de-activation (Figure 3O and P) in cells transfected with activated mDia1 but not in mDia1-untransfected cells (Figure 3K and L), cells transfected with autoinhibited mDia1 (Figure 3M and N), or RhoA(V14) alone (Figure 3Q and R). Similar data were obtained when K+ was used as the sole charge carrier ruling out the possibility of divalent-induced rectification patterns in I–V curves (Supplementary Figures S4 and S5). Overall, these data led us to propose that activated mDia1 conferred voltage sensitivity to overexpressed TRPP2 in LLC-PK1 cells and supported data on native LLC-PK1 cells. Structure–function analysis of mDia1 Next, we wished to provide a molecular basis for the voltage-dependent block of TRPP2 by mDia1 by identifying the minimal domain in mDia1 responsible for its effect on TRPP2 and also to confirm that activated constructs of mDia1 should confer voltage sensitivity to TRPP2 in the absence of activation through RhoA. Because TRPP2 showed higher conductance in K+ than Na+ or Ca2+ (Luo et al, 2003; Ma et al, 2005) and also to avoid Ca2+-induced rectifications, in these experiments we measured whole cell currents in symmetrical K+. A series of N- and C-terminal truncation mutants and the mDia1(M1182A) mutant, which was shown to render mDia1 in the activated state (Lammers et al, 2005), were tested for their effect on TRPP2. Transfection of the ΔN3mDia1 mutant, which lacked the TRPP2-binding site, was without effect (Figure 4A), whereas activated constructs mDia1(1–747) (Figure 4B), mDia1(1–1144) (Figure 4C), or mDia1(M1182A) (Figure 4D) conferred outward rectification on TRPP2 currents. mDia1(1–586) (Figure 4E) and mDia1(1–1203) (Figure 4F) behaved as wild-type mDia1 in the autoinhibited state. Construct mDia1(1–1169), which was expected to result in partial activation (Lammers et al, 2005), partially suppressed outward currents (Figure 4G). Tail current experiments in cells transfected with mDia1(1–586) and mDia1(1–1144) confirmed the voltage-dependent effect of activated mDia1 on TRPP2 currents (Supplementary Figure S6A–G). The voltage-dependent effect of activated forms of mDia1 such as mDia1(1–1144) was consistent with a Boltzmann distribution of mean conductance as a function of test potential (Figure 4H). Interestingly, partially activated mDia1(1–1169) showed a positive shift in V1/2 by about 28 mV (from 11.7±7.7 mV in mDia1(1–1144) to 39.8±2.7 mV) and significantly lower Gmax (from 10.0±0.1 nS in mDia1(1–1144) to 5.4±0.1 nS), indicating that the level of mDia1 activation dictated the voltage sensitivity of TRPP2. Summary data on structure-function analysis of mDia1 is shown in Figure 4I. Figure 4.Structure–function analysis of mDia1. (A–G) Pooled I–V curves of LLC-PK1TRPP2 cells transfected with ΔN3mDia1 (A), mDia1(1–747) (B), mDia1(1–1144) (C), mDia1(M1182A) (D), mDia1(1–586) (E), mDia1(1–1203) (F), or mDia1(1–1169) (G). Pooled I–V curve derived from untransfected LLC-PK1TRPP2 cells (Figure 3B) is shown in black for comparison (A–G). *P<0.05. (H) Boltzmann distributions of mean conductance (G) derived from steady-state currents at the end of the pulse as a function of test potential (Vm). (I) Summary data of structure–function analysis of mDia1. CC, coiled-coil region; DAD, diaphanous autoregulatory domain; DD, dimerization domain; DID, diaphanous inhibitory domain; G, GTPase-binding region; FH1, formin homology 1 domain; FH2, formin homology 2 domain; L143–L260, TRPP2-binding domain. Download figure Download PowerPoint Direct voltage-dependent gating of TRPP2 by mDia1 To determine whether the mDia1-dependent voltage gating of TRPP2 was direct, recombinant mDia1(1–586) or mDia1(1–747) (Figure 5A) was applied to the cytosolic side of inside-out patches excised from LLC-PK1TRPP2 cells. Time-course experiments indicated that 5 min starting perfusion of recombinant proteins was adequate to suppress TRPP2 inward currents (Figure 5B). Application of α-TRPP2 to the cytosolic side of excised patches of LLC-PK1TRPP2 cells confirmed the existence of functional TRPP2 by suppressing inward and outward macroscopic currents (Figure 5C–E). Application of normal rabbit IgG was without effect (Supplementary Figure S7A). Application of recombinant mDia1(1–586) suppressed macroscopic currents to levels identical to α-TRPP2 (Figure 5F–H) further confirming the voltage-independent action of this construct in excised patches. In sharp contrast, addition of recombinant mDia1(1–747) suppressed inward but not outward currents demonstrating a voltage-dependent effect of mDia1(1–747) on TRPP2 (Figure 5I–K). Consistently, mDia1(1–747) showed pronounced current relaxation and tail currents (Figure 5J) that were not seen in any of the patches incubated with α-TRPP2 or mDia1(1–586). It should be noted that the mDia1-dependent block of TRPP2 at negative potentials was irreversible for at least 5 min, following removal of recombinant mDia1 constructs from the bath (Supplementary Figure S7B–E). Figure 5.Voltage-dependent gating of TRPP2 by purified mDia1 in inside-out patches. (A) Coomassie blue staining of purified, recombinant MBP-mDia1(1–586) (lane 1), MBP-mDia1(1–747) (lane 2), or MBP (lane 3). (B) Time course of current inhibition by α-TRPP2 (2 μg/ml, n=3) (red) or recombinant MBP-mDia1(1–586) (7 μg/ml, n=3) (blue) applied directly to the bath solution in inside-out patches excised from LLC-PK1TRPP2 cells. Purified maltose-binding protein (MBP, 7 μg/ml, n=3) (green) or no protein (control, black) was used as negative controls. (C–E) Effect of bath application of α-TRPP2 on inside-out patches excised from LLC-PK1TRPP2 cells. Step currents before (C) and 5 min after (D) antibody application. (E) Pooled I–V curves before (black) and 5 min after (red) α-TRPP2 (2 μg/ml, n=3). (F–H) Effect of bath application of recombinant mDia1(1–586) on inside-out patches excised from LLC-PK1TRPP2 cells. Step currents before (F) and 5 min after (G) mDia1(1–586) addition in the bath. (H) Pooled I–V curves before (black) and 5 min after (red) mDia1(1–586) (7 μg/ml, n=6). (I–K) Effect of bath application of recombinant mDia1(1–747) on inside-out patches excised from LLC-PK1TRPP2 cells. Step currents before (I) and 5 min after (J) mDia1(1–747) addition in the bath. (K) Pooled I–V curves before (black) and 5 min after (red) mDia1(1–747) (10 μg/ml, n=5). *P<0.05. Download figure Download PowerPoint EGF-induced activation of TRPP2 through mDia1-dependent gating To determine the ph
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