LETTERS
TRPC1 forms the stretch-activated cation channel in vertebrate cells Rosario Maroto1, Albert Raso2, Thomas G. Wood3, Alex Kurosky3, Boris Martinac2 and Owen P. Hamill1,4 The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation1, cell locomotion2, muscle dystrophy3 and cardiac arrhythmias4. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp5. The oocyte was chosen because it expresses the prototypical MscCa (≥107 MscCa/oocyte)6 that is preserved in cytoskeleton-deficient membrane vesicles7. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1)8–10. Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels11. Measurements of the MscCa in cell-attached patches on frog oocytes indicate that there are few or no spontaneous openings, but that they can be experimentally activated if pressure or suction (~20 mmHg) is applied to the patch pipette12,13. Once activated, the channel displays a unitary chord conductance of ~40 pS (measured at –50 mV) in normal Ringer’s solution, and shows permeant ion block by divalent cations12–14. We find that this activity is preserved after oocyte membrane proteins have been solubilized in the detergent β-octylglucoside (OG) and reconstituted in liposomes. Figure 1a shows patch-clamp current recordings from an ‘inside-out’ isolated liposome patch at –80 mV in response to pressure
(~30 mmHg) applied to the pipette. In this case, the membrane proteins were reconstituted at a protein:lipid ratio of 1:100. The pressure pulse activated at least three unitary current events of ~2 pA. A low frequency (~1 s–1) of spontaneous current events of similar amplitude was recorded on the same patch (Fig. 1a, lower trace). Similar current events activated by pressures of 20–50 mmHg were recorded in 38 other patches formed on proteoliposomes of different composition (Figs 1b and 1d). By contrast, patches of pure liposomes failed to express unitary current events, even with applied pressures as high as 200 mmHg, which ultimately ruptured the patch/seal (five out of five patches tested). The single-channel current–voltage relationship that was measured under symmetrical 200 mM K+ with 40 mM internal Mg2+ (high Mg2+ was necessary to cause liposome blebbing, a critical requirement for patch-clamp recording5) indicated an inwardly rectifying channel (~30 pS at –50 mV and ~5 pS at 50 mV) that reversed at ~0 mV, and was similar to the MscCa recorded from inside-out oocyte patches under the same high internal Mg2+ ionic conditions (solid curve, Fig. 1b). Replacing external K+ with Cs+ did not significantly change inward currents, whereas inclusion of 40 mM Mg2+ in the external (pipette) solution significantly reduced inward conductance (~10 pS at –50 mV, Fig. 1b). These conductance properties and pressure sensitivity of the reconstituted channel were consistent with the properties of the oocyte MscCa6,7,12–14. To identify the specific protein(s) that may underlie the ‘MscCa-like’ activity, we carried out membrane-protein fractionation using fast performance liquid chromatography (FPLC) and obtained a protein profile with several distinct peaks. Fig. 1c shows a chromatogram of the observed protein elution pattern that was selected from chromatograms of three different frog preparations (see supplementary information, Fig. S1). Liposome reconstitution of the proteins representing several of the main peaks showed MscCa-like activity when reconstituted with a relatively high protein:lipid ratio (1:100; Fig. 1d). However, only one fraction (fraction 4*) retained activity when the ratio was reduced to 1:2000 or 1:5000. A similar peak MscCa active fraction, with a conductivity of 16 mS cm–1, was obtained from the two other frog oocyte preparations. A silver-stained gel of the fractionated proteins showed that the most active fraction displayed the highest abundance of a Mr 80K protein
1
Department of Neuroscience & Cell Biology, UTMB, Galveston, TX 77555, USA. 2School of Medicine and Pharmacology, University of Western Australia, Crawley, WA 6009, Australia. 3Department of Human Biological Chemistry & Genetics, UTMB, Galveston, TX 77555, USA. 4 Correspondence should be addressed to O.P.H. (e-mail:
[email protected]). Published online: 23 January 2005, DOI: 10.1038/ncb1218
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LETTERS b
c
1 pA
1 M NaCI
50 mmHg −120 40
2 pA
80 mV −1
Mg2+
−2
1s
−3 0
Mg2+ −4
Relative absorbance at 215 nm
a
1 2
4* 3
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Percentage of patches with MscCa
d
f Mr(K)
100
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97.4 -
TRCP1 antibody + BP
Mr 80K
66.2 -
80
20
Elution time (min)
e 1
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60 40
31.0 P:L ratio 1,100 1,2000
20 0
5
4
3 2 1 FPLC fraction
1,5000
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Figure 1 Mechanosensitive channel (MscCa) activity is preserved after protein detergent solubilization and liposome reconstitution of frog oocyte membrane. (a) Channel events recorded from an isolated proteoliposome patch composed of solubilized membrane proteins and azolectin phospholipids (ratio 1:100). The top trace shows the pressure recording during syringe-applied suction of ~30 mmHg. The middle trace shows the corresponding membrane patch current, with several current events of ~2 pA. The bottom trace shows the continued current trace with several spontaneous unitary current events. The pipette solution was 200 mM KCl, 5 mM Hepes and the bath solution (that is, facing the inside-out membrane) was 200 mM KCl, 40 mM MgCl2 and 5 mM Hepes. Pipette potential was –80 mV. (b) Current–voltage relationship of reconstituted stretch-activated channels with high (40) Mg2+ and in low (0) external Mg2+. In all cases, the
bath solution had high Mg2+ (200 mM K+ and 40 mM Mg2+). The solid curves represent fits to inside-out oocyte patch data based on four to five patches (data not shown to increase figure clarity) measured under the same ionic conditions as above. (c) Fast performance liquid chromatography (FPLC) profile measured for β-octylglucoside-solubilized oocyte membrane proteins using a UNO Q-1 anion-exchange column. (d) The percentage of patches showing MscCa activity, measured in proteoliposomes with different protein: lipid (P:L) ratios for proteins from the different FPLC fractions (five patches tested for each protein:lipid ratio). (e) Silver-stained gel of proteins collected from the fractions representing specific FPLC profile peaks run on a 12% SDS polyacrylamide gel. (f) Western blot of the proteins collected in fraction 4 tested with the TRPC1 antibody (1:200 dilution) and blockage shown by the antigenic peptide (blocking peptide, BP).
(Fig. 1e). We found the Mr 80K protein band to contain the canonical transient receptor potential channel 1 (TRPC1) using immunological techniques (Fig. 1f). The presence of TRPC1 was tested on the basis of earlier reports that TRPC1 cloned from Xenopus is expressed as a Mr 80K membrane protein in the oocyte cell membrane9,10. Previous studies indicated that TRPC1 formed a cation channel that only weakly discriminated between monovalent and divalent cations (reviewed in ref. 15). Although initially proposed to form the store-operated Ca2+ channel (SOCC) in oocytes9, subsequent studies failed to support this role10 and the exact function of TRPC1 remained unclear15. Because the human TRPC1 is highly homologous (84% identical and ~90% similar in amino acids) to the Xenopus TRPC1 (refs 8–10) and also results in expression of an apparent Mr 80K protein when expressed in oocytes10, we tested the effects of heterologous expression of human
TRPC1 on MscCa patch density in oocytes. Figure 2 compares currents in response to increasing pressure steps applied to a cell-attached patch (pipette solution: 100 mM KCl, 5 mM Hepes, 2 mM EGTA) on a control oocyte (water-injected; Fig. 2a, b) and an oocyte that has been injected 4 d previously with human TRPC1 transcripts (Fig. 2c, d). The latter showed a several-fold increase in the saturating currents (Fig. 2e), but the single-channel currents were similar (examples indicated by * in Figs a–d) and showed the same I–V relationship as the native MscCa (~50 pS at –50 mV, ~10 pS at 50 mV; Fig. 2f). Figure 3 shows the responses of another pair of patches to stepwise increases in pressure in which saturating currents of ~12 pA for the control (Fig. 3a) versus ~170 pA for the human TRPC1-transfected oocyte patch were evoked (Fig. 3b; note the pressure for half-maximum activation was ~20 mmHg in each case) and translated into MscCa patch densities of 5 (7 ±0.8, 30 patches, three frogs)
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LETTERS a
∗
b 50 mV
2 pA
5 pA
∗
−50 mV
20 pA
d Human TRPC1-expressing
c
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2.2 pA 30 pA 50 mV
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−1 − 150
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− 200 − 250
Figure 2 Stretch-activated multi-channel and single-channel currents measured in cell-attached patches from control (a, b) and human TRPC1expressing (c,d) oocytes at different patch potentials. (a) Current responses to increasing steps of suction at patch potentials of 50 and –50 mV. (b) Expanded region of a (that is, near *), indicating single-channel currents of 2 pA at –50 mV. (c) Similar to a, except that the patch was formed on an oocyte that was injected 4 d earlier with human TRPC1 transcripts. (d) Expanded region of trace in c, indicating single-channel currents of
~2 pA. (e) Current–voltage relationship of macroscopic currents from patches from control (three patches) and human TRPC1-injected oocytes (four patches). (f) Single-channel current–voltage relationship for channel currents measured in human TRPC-expressing oocytes, as indicated in d (two to four patches). The solid curve represents the best fit to singlechannel data recorded from the native oocyte mechanosensitive channel under similar conditions (based on 15 patches; data points not shown to increase figure clarity).
and 85 (98 ±17, 15 patches, two frogs) (Fig. 3e), respectively, assuming single-channel currents of ~2 pA (Figs. 3c, d). In comparison, injection of oocytes with antisense cRNA for human TRPC1 reduced endogenous MscCa activity (1.25 ±0.32, 20 patches, two frogs; Fig. 3e), along with the Mr 80K band that was measured using western blots (insert Fig. 3e). Gd3+, which blocks the oocyte MscCa13,16 and also TRPC channels15, was equally effective in blocking the native and the TRPC1-expressed MscCa (KD ~2 µM; see supplementary information, Fig. S2). To further test the idea that TRPC1 forms the MscCa, we screened several mammalian cell lines as possible ‘nulls’ for MscCa. However, of the seven cell lines tested, six (LMTK, Jurkat-T, HEK-293, BC3H-1, LNCaP and PC3) expressed relatively high MscCa activity (that is, with 70–100% patches active; R. Maroto and O.P. Hamill, unpublished observations),
compared with one (CHO-K1) that expressed low MscCa activity (that is, 10% active patches). Figure 4a shows a combined transmission and fluorescence photomicrograph of human TRPC1-transfected and non-transfected CHO-K1 cell colonies that are distinguished by their enhanced green fluorescent protein (eGFP) fluorescence. Figure 4b shows cell-attached patch recordings from human TRPC1-transfected and non-transfected cells in response to increasing steps of pressure and indicate the increased MscCa expression in human TRPC1-transfected (90% patches with 14 MscCa/active patch) versus non-transfected cells (10% patches with ≤2 MscCa/active patch) (Fig. 4c). Recordings indicate that both the native CHO-K1 and the human TRPC1-expressed MscCa channels (Fig. 4d) show a similar I–V relationship and Mg2+ sensitivity as the frog oocyte MscCa (Fig. 4e). These results are remarkable because
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a
Human TRPC1-expressing
Control oocyte 100 mmHg 80 mmHg
* * 25 pA 4s 400 ms
c
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Mr(K)
100
121 80
tis e
4s 400 ms
175 pA
d
An
e
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ntr o
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ns e
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Hu TR man ma PC1 nT an RPC tis en 1 se Sc ra an mbl tis ed en se Hu
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Figure 3 Mechanosensitive channel (MscCa) activity in control and human TRPC1 mRNA-injected oocytes and the effects of human TRPC antisense RNA on native MscCa activity. (a) Stepwise increase in suction (top trace) applied to a cell-attached patch on a control (water-injected) oocyte activates an inward current (lower trace) of ~12 pA. (b) Similar to a, except that the cell-attached patch was formed on an oocyte that was injected 4 d previously with human TRPC1 transcripts showing activation of ~175 pA current. (c) Expanded region near * in Fig. 1a shows residual single-channel current events of ~2 pA immediately after the pressure step. (d) Expanded region near * in Fig. 1b shows residual single-channel current events of ~2 pA immediately after the pressure step. For the cellattached patch recordings (Figs 1a—1d), the pipette solution contained 100 mM KCl, 2 mM EGTA (KOH) and 5 mM Hepes (KOH), and the
driving force was –20 mV (that is, measured from the reversal potential of ~ 0 mV of the MscCa channel currents). The two patch pipettes that were used for this comparison were pulled from the same capillary tube, thereby ensuring identical tips. (e) Histogram of MscCa/patch density measured in control (water-injected), human TRPC1 cRNA-injected, human TRPC1 antisense-cRNA-injected oocytes and scrambled antisenseoligonucleotide-injected oocytes (n = 30, 15, 20 and 10, respectively). Data plotted as mean (SEM ±*** p 20 GΩ) either formed immediately or after application of a brief pulse of negative pressure (
