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Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes
在非洲爪蟾卵母细胞中表达分析流量调节型的离子通道   

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Abstract

Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to examine the response of ion channels expressed in Xenopus oocytes to LSS (Hoger et al., 2002; Carattino et al., 2004; Shi et al., 2006). The Xenopus oocyte is a reliable system that allows for the expression and chemical modification of ion channels and regulatory proteins (George et al., 1989; Palmer et al., 1990; Sheng et al., 2001; Carattino et al., 2003). Therefore, this technique is suitable for studying the molecular mechanisms that allow flow-activated channels to respond to LSS.

Keywords: Flow(流量), Laminar shear stress(层流剪应力), Ion channels(离子通道), Xenopus oocytes(非洲爪蟾卵母细胞), Two-electrode voltage clamp(双电极电压钳), Microinjection(显微注射)

Background

Epithelial cells that line the urinary tract and endothelial cells that line blood vessels are subjected to mechanical forces elicited by moving fluids. These forces are, laminar shear stress (LSS), a frictional force tangential to the wall of the tubular structures, and circumferential stretch, which is perpendicular to the direction of flow. Compelling evidence indicates that LSS is the main determinant for the physiological responses observed in response to flow changes in tubular structures of the kidney and blood vessels (Satlin et al., 2001; Liu et al., 2003; Weinbaum et al., 2010). In these settings, ion channels have an important role transmitting fluid shear stress into biological signals (Ranade et al., 2015). For instance, in the distal nephron of the kidney the rates of Na+ reabsorption and K+ secretion are positively modulated by luminal fluid flow. In this segment of the nephron, high tubular flow rates enhance the activity of the epithelial sodium channel (ENaC) (Satlin et al., 2001 and 2006; Morimoto et al., 2006). In the face of high luminal flow rates, the apical entry of Na+ mediated by ENaC and its electrogenic basolateral extrusion create an electrochemical gradient that favors the passive diffusion of cellular K+ into the luminal fluid through maxi-K channels (Woda et al., 2001; Satlin et al., 2006). Likewise, in the vasculature, where fluid shear stress is essential for normal physiological responses, ion channels have been proposed as mechanosensors that mediate endothelial flow signaling (Davies, 1995; Hoger et al., 2002; Wang et al., 2009; Guo et al., 2016).

The technique described in this protocol has been used to examine basic aspects of the regulation of ENaC by LSS as well as to gain understanding of the molecular mechanisms that allow this channel to respond to fluid flow (Carattino et al., 2004; 2005 and 2007; Morimoto et al., 2006). With this technique, we were able to characterize basic features of the response of ENaC to LSS, such as time-course of activation, strain dependence, temperature dependence, and voltage dependence (Carattino et al., 2004 and 2007). In addition, using ENaC mutant subunits that assemble to form channels that are either constitutively open (βS518K) or that can be locked in an open state by chemical modification (αS580C), we showed that fluid flow increases ENaC activity by changing the open probability of the channel (Carattino et al., 2004). This finding was later confirmed using single channel analysis (Althaus et al., 2007). Moreover, by combining the technique described herein and site-directed mutagenesis we were able to identify key structural elements in ENaC required for a response to LSS (Carattino et al., 2004 and 2005; Abi-Antoun et al., 2011; Shi et al., 2011; 2012a; 2012b and 2013). Recently, we employed this technique to examine the regulation of MEC-4 and MEC-10 by LSS. These channel forming subunits are members of the ENaC/degenerin family expressed in C. elegans that are required for gentle touch in worms (Driscoll and Chalfie, 1991; Shi et al., 2016). Other investigators have employed the technique describe here to study the response of K+ channels expressed in the vasculature to LSS (Hoger et al., 2002; Fronius et al., 2010). In summary, the technique described in this protocol is suitable to examine the molecular mechanisms by which fluid flow regulates the function of epithelial and endothelial ion channels.

Materials and Reagents

  1. Oocyte injection Petri dish: a 10 cm diameter Petri dish (Fisher Scientific, FisherbrandTM, catalog number: FB0875713 ) with a polypropylene mesh of 60 μm opening size (Spectrum, catalog number: 146494 ) glued to the bottom
  2. 50 ml conical tubes (Corning, catalog number: 352098 )
  3. 15 ml conical tubes (Corning, catalog number: 352099 )
  4. Plastic transfer pipettes (Fisher Scientific, FisherbrandTM, catalog number: 13-711-9CM )
  5. Nuclease-free tubes (Eppendorf, catalog numbers: 022600001 and 022600028 )
  6. Nuclease-free tips (Mettler-Toledo, Rainin, catalog numbers: 17002927 and 17002928 )
  7. 3.5 in. glass capillaries (Drummond Scientific, catalog number: 3-000-203-G/X )
  8. Razor blade
  9. Syringe 5 ml (BD, catalog number: 309603 )
  10. Needles 25 G 1 1/2 (BD, catalog number: 305127 )
  11. Parafilm (Bemis, catalog number: PM992 )
  12. 6-well tissue culture plate (Corning, catalog number: 3516 )
  13. L-shaped capillary tube with an internal diameter of 1.8 mm (homemade)
  14. Glass capillaries 1.5 mm diameter (WPI, catalog number: 1B150F-4 )
  15. Tygon tubing 1/16 in ID 1/18 in OD (Fisher Scientific, FisherbrandTM, catalog number: 14-171-129 )
  16. Frogs, Xenopus laevis (Nasco, Fort Atkinson, WI)
  17. Tricaine methane sulfonate (MS-222) (Sigma-Aldrich, catalog number: C6885 )
  18. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6297 )
  19. Collagenase type II (Sigma-Aldrich, catalog number: C6885 )
  20. Soybean trypsin inhibitor (Sigma-Aldrich, catalog number: T9128 )
  21. Commercial in vitro transcription kit (e.g., mMessage mMachine transcription kits from Ambion)
  22. Nuclease free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
  23. Mineral oil (Sigma-Aldrich, catalog number: M3516 )
  24. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  26. HEPES (Sigma-Aldrich, catalog number: H3375 )
  27. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 30530 or S8045 )
    Note: The product “ 30530 ” has been discontinued.
  28. Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: C1396 )
  29. Calcium chloride dehydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  30. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643 )
  31. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  32. Sodium penicillin/streptomycin sulfate (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  33. Gentamycin sulfate (Thermo Fisher Scientific, GibcoTM, catalog number: 15750078 )
  34. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
  35. Benzamil hydrochloride (Sigma-Aldrich, catalog number: B2417 ): a potent blocker of ENaC and degenerin channels
  36. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8779 )
    Note: This product has been discontinued.
  37. Ca2+-free standard oocytes solution (SOS, see Recipes)
  38. Modified Barth solution (MBS) (see Recipes)
  39. Hypotonic solution (see Recipes)
  40. Two-electrode voltage clamp recording solution (TEV) (see Recipes)
  41. Benzamil working solution (see Recipes)

Equipment

  1. Harvesting of ovaries from female Xenopus laevis
    1. 2-L glass beaker
    2. Fine forceps for tissue collection (Roboz Surgical, catalog number: RS-5240 )
    3. Fine scissors for tissue collection (Fine Science Tools, catalog number: 14060-09 )
    4. Tissue forceps (Roboz Surgical, catalog number: RS-8162 )

  2. Oocyte isolation and maintenance
    1. Dumont size 4 forceps (Fine Science Tools, catalog number: 11241-30 )
    2. Clay AdamsTM Nutator mixer (BD, catalog number: 421105 )
    3. Oocytes transferring pipette: a polished glass Pasteur pipette (Fisher Scientific, FisherbrandTM, catalog number: 13-678-20 )
    4. Pipette pump (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F378980000 )
    5. B.O.D. low temperature refrigerated incubator (VWR)

  3. Oocyte injection
    1. Programmable nanoliter injector (Drummond Scientific, model: Nanoject III )
    2. Steel base plate (WPI, catalog number: 5052 )
    3. Magnetic holding stand (WPI, catalog number: M10 )
    4. Three-axis manual micromanipulator (WPI, catalog number: M3301 )
    5. Stereo microscope (Olympus, model: SZ61 )
    6. Fiber optic illuminator and bifurcated light guide (WPI)
    7. Micropipette pullers (NARISHIGE, model: PC10 )

  4. Two-electrode voltage clamp
    1. Steel base plate (WPI, catalog number: 5479 )
    2. Steel base plate (WPI, catalog number: 5052 )
    3. Magnetic holding devices, three (WPI, catalog number: M10 )
    4. Three-axis manual micromanipulators, three (WPI, catalog number: M3301 )
    5. Two-electrode voltage clamp amplifier (Molecular Devices, Axon Accessories, model: GeneClamp 500B )
    6. Headstages (Molecular Devices, Axon Accessories, model: HS-2A )
    7. Virtual ground bath clamp (Molecular Devices, Axon Accessories, model: VG-2A )
    8. Pipette holders (Molecular Devices, model: HL-U )
    9. Ag/AgCl pellets (2 mm diameter, 4 mm long) (WPI, catalog number: EP2 )
    10. Digitizer (Molecular Devices, model: Digidata 1550B )
    11. BNC cables (A-M systems)
    12. Perfusion system (Warner Instruments, model: VC-6 )
    13. Flow valve (Warner Instruments, model: FR-50 )
    14. Vacuum attached waste bottle (Fisher Scientific, FisherbrandTM, catalog number: FB3002000 )
    15. Oocyte chamber (20 mm diameter and 6 mm deep ring with glued glass bottom)
    16. Silver wire AWG 26 for electrodes (WPI, catalog number: AGW1510 )
    17. PC computer with Windows operating system

Software

  1. pClamp 10 software (Molecular Devices)

Procedure

  1. Harvesting of ovaries from female Xenopus laevis
    1. Anesthetize frogs in a 2-L beaker with anesthetic solution containing 0.15 % (w/v) tricaine methane sulfonate (MS-222) buffered with 0.2 % (w/v) NaHCO3 at room temperature. Frogs are immersed in the anesthetic solution (1 L) for at least 30 min or until lack of response to toe pinch (Figure 1A).
    2. Harvest ovaries through a lateral incision in the lower abdominal area. Avoid cutting the abdominal vein that runs through the center of the abdominal region, as it will result in unnecessary bleeding (see Figures 1B and 1C).
    3. Place harvested ovaries in a Petri dish containing ~20 ml MBS and incubate at 18 °C in B.O.D. incubator until further use.
    4. Euthanize the frog by immersion in the anesthetic solution for 1 h, followed by a thoracotomy and heart removal.


      Figure 1. Surgical removal of oocytes. A. Anesthesia depth is evaluated by pinching the toe with a pair of forceps. Surgical procedures are conducted when no withdrawal response to pinch is observed. B and C. Ovaries are harvested through a lateral incision in the lower abdominal area (B) using fine forceps (C).

  2. Oocytes isolation and sorting
    1. Prepare a collagenase stock (7x) solution by dissolving 21 mg of collagenase type IV and 50 mg of soybean trypsin inhibitor in 2 ml of Ca2+-free SOS.
    2. Gently dissociate ovarian lobes in small pieces (0.5-1 cm diameter) with a pair of Dumont size 4 forceps in a Petri dish containing MBS.
    3. Transfer ovary fragments to a 50 ml conical tube and wash 3 times with 50 ml of Ca2+-free SOS. To wash the fragments, leave the tube in vertical position until the fragments sink to the bottom, then gently rotate the tube to dump the solution covering the pieces of tissue.
    4. Transfer ~7 ml of washed ovary fragments to a 15 ml conical tube containing 3 ml of Ca2+-free SOS.
    5. Add 2 ml of collagenase stock solution to the 15 ml conical tube containing the ovary fragments, fill the tube with Ca2+-free SOS to 14 ml and close the tube.
    6. Rotate the tubes containing ovary pieces in the Nutator Mixer at 12 rpm for one hour at room temperature.
    7. After 1 h of incubation, use a plastic transfer pipette with a cut tip (~5 mm diameter) to transfer an aliquot of the digest to a Petri dish. Oocyte should be submerged in the solution and not come in contact with air at any time. Visually inspect the oocyte under a stereomicroscope. The digestion with collagenase is completed when oocytes lack most of the extracellular connective tissues and vessels (see Figure 2C). If after 1 h incubation a significant amount of connective tissue and vessels are still visible, extend the incubation for 10 min more, or as required. Take into consideration that excess of collagenase digestion can affect the viability of the oocytes.
    8. To remove remaining pieces of connective tissue that might still attached to the oocytes after the collagenase digestion, transfer the oocytes (and solution) from the 15 ml conical tube to a 50 ml conical tube. Leave the tube in vertical position until the oocytes sink to the bottom, then gently rotate the tube and dump the solution covering the oocytes. Fill the tube with 30 ml of hypotonic solution, mix gently and dump the solution covering the oocytes. Repeat the last step twice.
    9. Incubate the oocytes with 30 ml of hypotonic solution for 15 min at room temperature on a bench (without agitation).
    10. After the incubation, wash the oocytes three times with MBS and place them in a Petri dish containing MBS. Store the oocytes in the B.O.D. incubator at 18 °C for at least two hours before proceeding to sorting.
    11. Select oocyte stage V and VI with the oocytes transferring pipette (see Figure 2E). At these stages of development, oocytes normally have a well-defined belt separating the animal pole (dark) from the vegetal pole (light).


      Figure 2. Isolation of Xenopus laevis oocytes. A. Macroscopic view of harvested ovaries; B. Close view of an ovary sac; C. Close view of oocytes after collagenase digestion. Note that some connective tissue and vessels still present in the surface of the oocytes. D. Close view of oocytes after treatment with the hypotonic solution; E. Close view of oocytes at development stages 1-6. Note that stage 5-6 oocytes have a diameter of approximately 1 mm. 

  3. Oocyte injection
    1. cRNA is produced with a commercial in vitro transcription kit following the supplier directions. We use mMessage mMachine transcription kits from Ambion. Use nuclease free water and nuclease-free tubes and tips to prepare and handle cRNA solutions.
    2. Pull injection needles from 3.5” glass capillaries using a single step protocol (one step, heater level 65.1). Cut the tip to a 30° angle with a razor blade. The diameter of the tip of the injection needles should be approximately 50 µm.
      Note: Injection needles with angled fine tips enable wound healing and increase oocytes survival.
    3. Set the injection volume at 50 nl and the injection speed at fast. Fill the injection pipette with mineral oil using a syringe with a 25 G needle. Plug the filled injection pipette in the microinjector and push the mineral oil out with the ‘empty’ bottom (Figure 3A).


      Figure 3. Oocytes injection workstation. A. Injection pipettes made of glass capillaries are used for injection of cRNAs encoding for ion channels and other proteins in Xenopus oocytes. Injection workstation components labeled as follows: 1. Stereo microscope with external light source; 2. Micromanipulator and magnetic holding device; 3. Nanoinjector; 4. Nanoinjector controller. B. Loading of injection needle with cRNA. The injection needle is filled with mineral oil. A drop of the solution containing the cRNA of interest is place in the center of a piece of Parafilm. cRNA solution is collect with the injection pipette using the ‘fill’ bottom in the nanoinjector controller. C. Close view of oocytes aligned in a Petri dish. To prevent rolling during injection, oocytes are placed in a Petri dish with a polypropylene mesh glued to the bottom.

    4. Place a square piece of Parafilm (~2 x 2 cm) in the field of view of the stereomicroscope (Figure 3B). Place a drop of the solution containing the cRNA of interest (5-6 µl) in the center of the Parafilm. Collect the cRNA solution with the injection pipette using the ‘fill’ bottom. Press the injection bottom several times to verify that the injection pipette is working properly.
    5. Fill the oocyte injection Petri dish with MBS. Transfer 20-40 oocytes from the incubation dish with a polished Pasteur pipet and arrange them in a line in the injection Petri dish (Figure 3C). Move the injection pipette close to the oocytes.
    6. Gently impale an oocyte in the center of the animal pole (dark side) and inject the cRNA by pressing the ‘inject’ bottom. Hold the injection pipette for 3-4 sec inside the oocyte. Retract the injection pipette and sequentially inject the next oocyte in the line.
    7. Collect the injected oocytes and place them in a 6-well tissue culture plate filled with MBS. Incubate the oocytes at 18 °C for at least 20 h. The incubation time required for expression depends on the ion channel being studied.
      Note: For expression of ENaC, 1-2 ng cRNA/subunit is routinely injected and 24-48 h of incubation are required. For other channels, such as C. elegans degenerin channels, 5-10 ng cRNA/subunit are injected and a longer incubation (> 4 days) is required for optimal expression.

  4. Flow apparatus and estimation of laminar shear stress rate
    1. A software-controlled gravity-fed inflow perfusion system and vacuum-assisted outflow is utilized to perfuse the chamber used for electrophysiological recordings (Figure 4). The apparatus employed to deliver the fluid-shearing flow consist of an L-shaped glass pipette with an inner diameter of 1.8 mm, which is connected to one channel of the perfusion system. The fluid-shearing pipette is made of a 1.8 inner diameter capillary glass. The capillary glass is bent to L-shape (~ 90° angle) with a Bunsen burner. The fluid-shearing pipette is mounted in a three-axis manual micromanipulator in front of the recording chamber (see Figure 5B).


      Figure 4. Schematic of the recording chamber and perfusion system used to study flow-regulated ion channels. A home-made (20-mm diameter and 6-mm deep) chamber is used to bath the oocytes. To apply laminar shear stress, a homemade vertical perfusion glass tube (1.8 mm internal diameter) is placed right above the oocytes. Fluid is delivered through the fluid-shearing pipette using a software-controlled gravity perfusion system.


      Figure 5. Two-electrode-voltage (TEV) clamp of Xenopus oocytes. View of the two-electrode voltage clamp rig. A. Components are labeled as follows: 1. GeneClamp 500B amplifier and Digidata 1550B; 2. VC-6 PTFE valve perfusion system; 3. Stereo microscope with external light source; 4. Dell OPTIPLEX desktop; 5. Left and right electrode holders, micromanipulators and magnetic holding devices; 6. Fluid-shearing pipette, micromanipulator and magnetic holding device; 7. Flow valve. B. Close view of the oocyte flow chamber and perfusion system.

    2. The rate of laminar shear stress (LSS) on the oocyte surface is estimated as the relation of the drag force (Fdrag) and the surface area of the oocyte, LSS = Fdrag/surface area of the oocyte. The magnitude of the Fdrag is determined according to Fdrag = 0.5 ρAω2Cd, where ρ represents the water density (1 g/cm3), A is the cross-sectional area of the sphere (πr2) (for Xenopus oocytes r = 0.05 cm), ω is the average free stream velocity (flow rate/cross sectional area of the perfusion pipette) and Cd is the drag coefficient (~1 for spheres with a Reynolds number within the 3-25 range). The Reynolds number is determined as, Re = θωD/λ, where θ is the fluid density, ω is the average free stream velocity, D is the sphere diameter and λ is the viscosity of the water. For example, a perfusion rate of 1.5 ml/min is needed to generate a shear stress of 0.12 dynes/cm2 with a fluid-shearing pipette with a diameter of 1.8 mm.

  5. Two-electrode voltage clamp
    1. Experiments are conducted at room temperature (20-25 °C) on a two-electrode voltage clamp rig as shown in Figure 5.
    2. Estimate required flow rate to achieve desired rate of LSS as indicated in step D2.
    3. Fill the perfusion syringes with TEV solution. Adjust the perfusion rate of the bath solution to ~3 ml/min. Adjust the flow of the fluid-shearing pipette to the desired rate with the flow valve. The flow rate is determined by gravimetry by collecting fluid for a minute in a pre-weighted tube. Alternatively, a flowmeter can be connected in line with the perfusion system to measure the flow rate.
    4. Pull recording pipettes from 4” glass capillaries (1.5 mm) using a two-step protocol with the micropipette puller (set heater level step 1 at 47.2 and step 2 at 62.1). Backfill glass pipettes with 3 M KCl and gently tap them to release air bubbles from the tips.
    5. Transfer an injected oocyte to the center of the recording chamber (see Figure 5B).
    6. Mount the recording pipettes onto the left and right electrode holders. With the amplifier in the setup mode, carefully place the recording pipettes in the chamber to measure their resistance in the TEV solution.
      Note: The resistances of the pipettes should be between 0.2-2 mΩ. Pipettes with resistances < 0.1 mΩ are considered leaky and should be replaced.
    7. Adjust the offset of the recording electrodes to zero.
    8. Move the recording electrodes close to the oocyte by altering the X-Y-Z knobs of the micromanipulator. Gently impale the oocyte with the recording electrodes using the Z knob.
    9. The fluid-shearing pipette should be visible with the stereo microscope. Using the stereo microscope, position the tip of the fluid-shearing pipette directly above the oocyte. Carefully move the fluid-shearing pipette down within 0.5-1.0 mm of top of the oocyte.
    10. Set the amplifier to voltage-clamp mode.
    11. For ENaC, currents are continuously recorded at a membrane potential at -60 mV. The perfusion system is controlled with external TTL signals using pClamp 10. During the recordings, the chamber is continuously perfused with TEV solution. Pulse of fluid are applied through the fluid-shearing pipette. Usually, we record currents with bath perfusion for the first 30 sec, we apply fluid shear stress for 60 sec or until the current plateaus, and then we perfuse the chamber with a TEV solution containing benzamil (5 μM) for 30 sec to determine the leak current.
    12. To examine the voltage-dependence of the flow-activated component, whole cell currents are measured using a series of voltage steps (e.g., 500 msec) from -140 to 60 mV under basal conditions, in the presence of fluid shear stress and after perfusion with a channel inhibitor (see Carattino et al., 2006 for details).
    13. At the end of the experiment, move the fluid-shearing pipette to the initial position, remove the recording pipettes from the oocytes and discard the oocyte.
      Note: Benzamil or other channel blockers used during the experiments must be washed off by flushing TEV solution in the bath and vertical tube for at least 3 min.

Data analysis

  1. For ENaC and MEC channels, the magnitude of response to LSS (LSS response) is defined as the ratio of the benzamil-sensitive component of the current in the presence of LSS (I¬+LSS) and the benzamil-sensitive component of the current under basal conditions (I¬-LSS) (see Figure 6). I¬-LSS is determined by subtracting the benzamil-insensitive current (Ibenz), determined in the presence of benzamil in the bath, to the current measured just prior to the initiation of LSS (Ibasal). I¬+LSS is calculated by subtracting the benzamil-insensitive current to the peak current evoked by LSS (Iflow).


    Figure 6. Flow-mediated activation of C. elegans MEC-4/MEC-10 channels in Xenopus oocytes. Oocytes were injected with cRNAs encoding for MEC-4/MEC-10. LSS (0.12 dynes/cm2) was applied thorough the fluid-shearing pipette with continuous bath perfusion. The membrane potential was clamped at -60 mV. Vertical flow of 1.5 ml/min (grey bar) was initiated at t = 30 sec. At the end of experiment, TEV solution containing benzamil (5 μM) was perfused through the bath (black bar). Three measurements were made: the baseline current (Ibasal), LSS-stimulated current (Iflow) and benzamil-insensitive current (Ibenz). The magnitude of the response of MEC-4/MEC-10 channels to LSS (LSS response) was estimated as I¬+LSS/I¬-LSS.

  2. Time constants for channel activation (τ) are determined by fitting the first 60 sec of current increase following the initiation of vertical perfusion to an exponential equation:

        I = c + a × e(-t/τ)

    where,
    I is the macroscopic current,
    τ is the time constant,
    a and c are constants determined by curve fitting.

Notes

There is an inherent variability in the expression of proteins in Xenopus oocytes, which results from seasonal variation in the quality of the oocytes, extent of collagenase treatment and the cRNA injection procedure. To account for the variability in the expression, experiments are repeated with several batches of oocytes harvested from different frogs.

Recipes

  1. Ca2+-free standard oocyte solution (SOS), RT
    100 mM NaCl
    2 mM KCl
    5 mM HEPES
    Adjust pH to 7.4 using 1 N NaOH
    Store at RT (Room temperature)
  2. Modified Barth solution (MBS)
    88 mM NaCl
    1 mM KCl
    24 mM NaHCO3
    15 mM HEPES
    0.3 mM Ca(NO3)2
    0.41 mM CaCl2
    0.82 mM MgSO4
    10 μg/ml sodium penicillin
    10 μg/ml streptomycin sulfate
    100 μg/ml gentamycin sulfate
    Adjust pH to 7.4 using 1 N NaOH
    Store at 18 °C
  3. Hypotonic solution
    100 mM K2HPO4
    1 g/L BSA
    Adjust pH to 6.5
    Store at RT
  4. Two-electrode voltage clamp recording solution (TEV)
    110 mM NaCl
    2 mM KCl
    1.54 mM CaCl2
    10 mM HEPES
    Adjust pH to 7.4 using 1 N NaOH
    Store at RT
  5. Benzamil working solution
    Make 0.1 M benzamil stock in DMSO and keep in dark
    Dilute to final concentration with TEV solution (see Recipe 4) freshly on the day of experiment

Acknowledgments

This work was supported by NIH grants R01DK084060 (M.D.C.), K01DK103834 (S.S.), and by the Cellular Physiology and Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30-DK079307).

References

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  15. Palmer, L. G., Corthesy-Theulaz, I., Gaeggeler, H. P., Kraehenbuhl, J. P. and Rossier, B. (1990). Expression of epithelial Na channels in Xenopus oocytes. J Gen Physiol 96(1): 23-46.
  16. Ranade, S. S., Syeda, R. and Patapoutian, A. (2015). Mechanically activated ion channels. Neuron 87(6): 1162-1179.
  17. Satlin, L. M., Carattino, M. D., Liu, W. and Kleyman, T. R. (2006). Regulation of cation transport in the distal nephron by mechanical forces. Am J Physiol Renal Physiol 291(5): F923-931.
  18. Satlin, L. M., Sheng, S., Woda, C. B. and Kleyman, T. R. (2001). Epithelial Na+ channels are regulated by flow. Am J Physiol Renal Physiol 280(6): F1010-1018.
  19. Sheng, S., Li, J., McNulty, K. A., Kieber-Emmons, T. and Kleyman, T. R. (2001). Epithelial sodium channel pore region. Structure and role in gating. J Biol Chem 276(2): 1326-1334.
  20. Shi, S., Blobner, B. M., Kashlan, O. B. and Kleyman, T. R. (2012a). Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress. J Biol Chem 287(19): 15439-15444.
  21. Shi, S., Carattino, M. D., Hughey, R. P. and Kleyman, T. R. (2013). ENaC regulation by proteases and shear stress. Curr Mol Pharmacol 6(1): 28-34.
  22. Shi, S., Carattino, M. D. and Kleyman, T. R. (2012b). Role of the wrist domain in the response of the epithelial sodium channel to external stimuli. J Biol Chem 287(53): 44027-44035.
  23. Shi, S., Ghosh, D. D., Okumura, S., Carattino, M. D., Kashlan, O. B., Sheng, S. and Kleyman, T. R. (2011). Base of the thumb domain modulates epithelial sodium channel gating. J Biol Chem 286(17): 14753-14761.
  24. Shi, S., Luke, C. J., Miedel, M. T., Silverman, G. A. and Kleyman, T. R. (2016). Activation of the Caenorhabditis elegans degenerin channel by shear stress requires the MEC-10 subunit. J Biol Chem 291(27): 14012-14022.
  25. Wang, S., Meng, F., Mohan, S., Champaneri, B. and Gu, Y. (2009). Functional ENaC channels expressed in endothelial cells: a new candidate for mediating shear force. Microcirculation 16(3): 276-287.
  26. Weinbaum, S., Duan, Y., Satlin, L. M., Wang, T. and Weinstein, A. M. (2010). Mechanotransduction in the renal tubule. Am J Physiol Renal Physiol 299(6): F1220-1236.
  27. Woda, C. B., Bragin, A., Kleyman, T. R. and Satlin, L. M. (2001). Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 280(5): F786-793.

简介

机械门控离子通道在机械传导中起关键作用,这是将物理力量转化为生物信号的过程。 上皮细胞和内皮细胞暴露于层流剪切应力(LSS),这是通过流体流向血管壁和上皮细胞壁所施加的切向力。 本文概述的方案已用于检查在非洲爪蟾卵母细胞中表达的离子通道对LSS的反应(Hoger等,2002; Carattino等,2004; Shi等,2006)。 非洲爪蟾卵母细胞是允许离子通道和调节蛋白的表达和化学修饰的可靠系统(George等,1989; Palmer等,1990; Sheng等,2001; Carattino等,2003)。 因此,该技术适用于研究允许流激活通道响应LSS的分子机制。
【背景】排列血管的泌尿道和内皮细胞的上皮细胞经受移动流体引起的机械力。这些力是层流剪切应力(LSS),与管状结构壁相切的摩擦力,以及垂直于流动方向的周向拉伸。令人信服的证据表明,LSS是响应肾和血管管状结构的流动变化而观察到的生理反应的主要决定因素(Satlin等,2001; Liu等,2003; Weinbaum等,2010) 。在这些设置中,离子通道具有将流体剪切应力传递到生物信号中的重要作用(Ranade等,2015)。例如,在肾的远端肾单位中,Na +再吸收和K +分泌的速率通过管腔流体流积极调节。在肾单位的这一部分,高管状流速增加了上皮钠通道(ENaC)的活性(Satlin等,2001和2006; Morimoto等,2006)。面对高腔体流速,由ENaC介导的Na +及其电生物基底外侧挤压引起的顶端进入产生电化学梯度,有利于细胞K +被通过最大K通道的腔内液体的被动扩散(Woda et al。,2001 ; Satlin等,2006)。同样,在脉管系统中,流体剪切应力对于正常的生理反应是必需的,离子通道已经被提出作为介导内皮细胞信号传导的机械传感器(Davies,1995; Hoger等,2002; Wang et al。,2009; Guo et等等,2016)。
本协议中描述的技术已被用于检查LSS对ENaC调节的基本方面,并且了解允许该通道对流体流动作出响应的分子机制(Carattino等,2004; 2005和2007 ; Morimoto等人,2006)。通过这种技术,我们能够表征ENaC对LSS响应的基本特征,如激活时间过程,应变依赖性,温度依赖性和电压依赖性(Carattino等,2004和2007)。此外,使用组装形成通道(组成型开放(βS518K))或可通过化学修饰(αS580C)锁定在开放状态的ENaC突变体亚基,我们显示流体流动通过改变开放概率来增加ENaC活性通道(Carattino等,2004)。随后使用单通道分析确认了这一发现(Althaus等,2007)。此外,通过结合本文描述的技术和定点诱变,我们能够鉴定对LSS的响应所需的ENaC中的关键结构元件(Carattino等人,2004和2005; Abi-Antoun等人,2011; Shi et 2011; 2012a; 2012b和2013)。最近,我们采用这种技术来检查LSS对MEC-4和MEC-10的调控。这些通道形成亚基是在线虫中表达的ENaC /简并蛋白家族的成员,其是蠕虫中温和触感所需要的(Driscoll和Chalfie,1991; Shi等人,2016)。其他研究者已经采用这里描述的技术来研究在脉管系统中表达的K +通道对LSS的反应(Hoger等,2002; Fronius等,2010)。总之,本方案中描述的技术适用于检查流体流动调节上皮和内皮离子通道功能的分子机制。

关键字:流量, 层流剪应力, 离子通道, 非洲爪蟾卵母细胞, 双电极电压钳, 显微注射

材料和试剂

  1. 卵母细胞注射培养皿:将具有60μm开口尺寸的聚丙烯筛网(Spectrum,目录号:146494)的10cm直径培养皿(Fisher Scientific,Fisherbrand TM,目录号:FB0875713)粘合到底部
  2. 50ml锥形管(Corning,目录号:352098)
  3. 15ml锥形管(Corning,目录号:352099)
  4. 塑料输送移液器(Fisher Scientific,Fisherbrand TM ,目录号:13-711-9CM)
  5. 无核酸管(Eppendorf,目录号:022600001和022600028)
  6. 无核酸酶的提示(Mettler-Toledo,Rainin,目录号:17002927和17002928)
  7. 3.5英寸玻璃毛细管(Drummond Scientific,目录号:3-000-203-G/X)
  8. 剃刀刀片
  9. 注射器5 ml(BD,目录号:309603)
  10. 针25 G 1 1/2(BD,目录号:305127)
  11. 石蜡膜(Bemis,目录号:PM992)
  12. 6孔组织培养板(Corning,目录号:3516)
  13. 内径为1.8毫米(自制)的L形毛细管
  14. 玻璃毛细管直径1.5毫米(WPI,目录号:1B150F-4)
  15. Tygon管1/16(ID 1/18 in OD)(Fisher Scientific,Fisherbrand TM ,目录号:14-171-129)
  16. 青蛙,非洲爪蟾(Necco,Fort Atkinson,WI)
  17. 甲磺酸三甲酯(MS-222)(Sigma-Aldrich,目录号:C6885)
  18. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S6297)
  19. 胶原酶II型(Sigma-Aldrich,目录号:C6885)
  20. 大豆胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T9128)
  21. 转录试剂盒(例如,Ambion的mMessage mMachine转录试剂盒)
  22. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9937)
  23. 矿物油(Sigma-Aldrich,目录号:M3516)
  24. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  26. HEPES(Sigma-Aldrich,目录号:H3375)
  27. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:30530或S8045)
    注意:产品"30530"已经停产。
  28. 硝酸钙四水合物,Ca(NO 3 3)2·4H 2 O(Sigma-Aldrich,目录号:C1396)
  29. 氯化钙脱水(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  30. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M2643)
  31. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  32. 青霉素钠/硫酸链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
  33. 硫酸庆大霉素(Thermo Fisher Scientific,Gibco TM,目录号:15750078)
  34. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
  35. 苯甲酸盐酸盐(Sigma-Aldrich,目录号:B2417):ENaC和退行性通道的有效阻断剂
  36. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8779)
    注意:本产品已停产。
  37. Ca 2 + - 标准卵母细胞溶液(SOS,参见食谱)
  38. 改良巴斯溶液(MBS)(见食谱)
  39. 低音解决方案(请参阅食谱)
  40. 双电极电压钳记录解决方案(TEV)(见配方)
  41. Benzamil工作解决方案(参见食谱)

设备

  1. 从非洲爪蟾女性收获卵巢的卵巢
    1. 2升玻璃烧杯
    2. 用于组织收集的精细镊子(Roboz Surgical,目录号:RS-5240)
    3. 精细剪刀用于组织收集(Fine Science Tools,目录号:14060-09)
    4. 组织钳(Roboz Surgical,目录号:RS-8162)

  2. 卵母细胞分离和维护
    1. Dumont尺寸4镊子(精细科学工具,目录号:11241-30)
    2. 粘土亚当斯 TM 营养搅拌机(BD,目录号:421105)
    3. 卵母细胞转移移液管:抛光玻璃巴斯德吸管(Fisher Scientific,Fisherbrand TM ,目录号:13-678-20)
    4. 移液泵(SP科学软件 - 贝尔艺术产品 - H-B仪器,目录号:F378980000)
    5. B.O.D.低温冷藏培养箱(VWR)

  3. 卵母细胞注射
    1. 可编程纳升喷射器(Drummond Scientific,型号:Nanoject III)
    2. 钢底板(WPI,目录号:5052)
    3. 磁力支架(WPI,目录号:M10)
    4. 三轴手动显微操纵器(WPI,目录号:M3301)
    5. 立体显微镜(Olympus,型号:SZ61)
    6. 光纤照明器和分支光导(WPI)
    7. 微量拔取器(NARISHIGE,型号:PC10)

  4. 双电极电压钳
    1. 钢底板(WPI,目录号:5479)
    2. 钢底板(WPI,目录号:5052)
    3. 磁保持装置,三(WPI,目录号:M10)
    4. 三轴手动显微操纵器,三(WPI,目录号:M3301)
    5. 双极电压钳位放大器(Molecular Devices,Axon Accessories,型号:GeneClamp 500B)
    6. Headstages(分子装置,Axon配件,型号:HS-2A)
    7. 虚拟地面浴夹(Molecular Devices,Axon Accessories,型号:VG-2A)
    8. 移液器支架(Molecular Devices,型号:HL-U)
    9. Ag/AgCl颗粒(2mm直径,4mm长)(WPI,目录号:EP2)
    10. 数字化仪(Molecular Devices,型号:Digidata 1550B)
    11. BNC电缆(A-M系统)
    12. 灌注系统(华纳仪器,型号:VC-6)
    13. 流量阀(Warner Instruments,型号:FR-50)
    14. 真空附加废物瓶(Fisher Scientific,Fisherbrand TM ,目录号:FB3002000)
    15. 卵母细胞室(20毫米直径和6毫米深的环,胶合玻璃底部)
    16. 用于电极的银线AWG 26(WPI,目录号:AGW1510)
    17. 带Windows操作系统的PC电脑

软件

  1. pClamp 10软件(Molecular Devices)

程序

  1. 从非洲爪蟾女性收获卵巢的卵巢
    1. 在室温下用含0.2%(w/v)NaHCO 3缓冲的0.15%(w/v)三烯丙基甲磺酸盐(MS-222)的麻醉溶液麻醉2升烧杯中的青蛙。将青蛙浸入麻醉溶液(1升)至少30分钟,或直到对脚趾夹紧的反应不足(图1A)。
    2. 通过下腹部的侧切口收获卵巢。避免切开穿过腹部中心的腹部静脉,否则会导致不必要的出血(见图1B和1C)。
    3. 将收获的卵巢置于含有约20ml MBS的培养皿中,并在18℃在B.O.D中孵育。孵化器直至进一步使用。
    4. 通过浸入麻醉溶液中将青蛙安乐死1小时,然后进行开胸手术和心脏移除。


      图1.手术切除卵母细胞。 A.通过用一对镊子捏住脚趾来评估麻醉深度。当不观察到捏合的撤回反应时,进行外科手术。 B和C.卵巢通过使用细镊子(C)的下腹部区域(B)的侧切口收获。

  2. 卵母细胞分离和分选
    1. 通过将21mg胶原酶IV和50mg大豆胰蛋白酶抑制剂溶解在2ml不含Ca 2+的无SOS中来制备胶原酶原料(7x)溶液。
    2. 在含有MBS的培养皿中用一对Dumont大小4镊子轻轻地将卵巢瓣分开(0.5-1厘米直径)。
    3. 将卵巢碎片转移到50ml锥形管中,并用50ml无Ca 2+的SOS洗涤3次。为了清洗碎片,将管子放在垂直位置,直到碎片沉入底部,然后轻轻地旋转管子以将覆盖在纸巾上的溶液倾倒。
    4. 将约7ml经洗涤的卵巢片段转移到含有3ml无Ca 2+的15ml锥形管中。
    5. 向含有卵巢片段的15ml锥形管中加入2ml胶原酶储备溶液,将管中的Ca 2 +无免疫SOS填充至14ml,并关闭管。
    6. 在Nutr搅拌机中以12rpm将包含卵巢片的管在室温下旋转1小时。
    7. 孵育1小时后,使用具有切割尖端(约5毫米直径)的塑料转移移液管将消化液的等分试样转移到培养皿中。卵母细胞应该浸没在溶液中,不要随时与空气接触。在立体显微镜下目视检查卵母细胞。当卵母细胞缺乏大部分细胞外结缔组织和血管时,胶原酶的消化完成(见图2C)。如果孵化1小时后,显着量的结缔组织和血管仍然可见,延长孵化10分钟以上,或根据需要。考虑到胶原酶消化过多会影响卵母细胞的生存能力
    8. 为了去除胶原酶消化后可能仍然附着于卵母细胞的剩余的结缔组织,将卵母细胞(和溶液)从15ml锥形管转移到50ml锥形管中。将管置于垂直位置,直到卵母细胞沉入底部,然后轻轻旋转管并将覆盖卵母细胞的溶液倾倒。向管中注入30ml低渗溶液,轻轻混匀,倒出覆盖卵母细胞的溶液。重复最后一步两次。
    9. 将卵母细胞与室温下30毫升低渗溶液孵育15分钟(不搅拌)。
    10. 孵育后,用MBS洗涤卵母细胞三次,并将其置于含有MBS的培养皿中。将卵母细胞储存在B.O.D.培养箱在18℃至少2小时,然后进行分选。
    11. 用卵母细胞转移移液管选择卵母细胞阶段V和VI(见图2E)。在这些发展阶段,卵母细胞一般都有一个明确的皮带,将动物极(黑暗)与植物极(光)分开。


      图2.分离非洲爪蟾卵母细胞 A.收获卵巢的宏观视图; B.仔细观察卵巢囊; C.胶原酶消化后观察卵母细胞。请注意,一些结缔组织和血管仍然存在于卵母细胞的表面。 D.用低渗溶液处理后仔细观察卵母细胞; E.在发育阶段1-6关闭卵母细胞的观察。请注意,阶段5-6卵母细胞的直径约为1mm。 

  3. 卵母细胞注射
    1. 根据供应商指导,使用商业化的体外转录试剂盒生产cRNA。我们使用Ambion的mMessage mMachine转录试剂盒。使用无核酸酶的水和无核酸酶的管和提示来制备和处理cRNA溶液。
    2. 使用单步协议从3.5"玻璃毛细管拉出注射针(一步,加热器等级65.1)。用刀片将尖端切割成30°角。注射针尖端的直径应为约50μm。
      注意:具有倾斜精细尖端的注射针使伤口愈合并增加卵母细胞存活。
    3. 注射体积设定在50 nl,注射速度快。使用25 G针头的注射器将注射器吸取矿物油。将填充的注射吸管插入微量注射器,并用"空"底部推出矿物油(图3A)。


      图3.卵母细胞注射工作站。A.由玻璃毛细管制成的注射移液管用于注射编码非洲爪蟾卵母细胞中的离子通道和其他蛋白质的cRNA。注射工作站部件标示如下:1.立体显微镜带外部光源;微操纵器和磁力保持装置;纳米注射器纳米注射器控制器B.用cRNA装入注射针。注射针充满矿物油。一滴含有感兴趣的cRNA的溶液位于一片Parafilm的中心。用纳米注射器控制器中的"填充"底部用注射吸管收集cRNA溶液。 C.仔细观察培养皿中排卵的卵母细胞。为了防止注射过程中的滚动,将卵母细胞置于带有粘合在底部的聚丙烯网的培养皿中
    4. 在立体显微镜的视野中放置一块方形的Parafilm(〜2 x 2厘米)(图3B)。将一滴含有感兴趣的cRNA(5-6μl)的溶液置于Parafilm的中心。使用"填充"底部的注射移液管收集cRNA溶液。按下注射底部几次以验证注射移液器是否正常工作。
    5. 填充卵母细胞注射培养皿与MBS。用抛光的巴斯德吸管从培养皿中转移20-40个卵母细胞,并将它们排列在注射培养皿中的一条线上(图3C)。将注射器移至靠近卵母细胞。
    6. 轻轻地将卵母细胞浸入动物杆的中心(黑暗面),然后按下"注射"底部注射cRNA。在卵母细胞内持续注射移液管3-4秒。撤回注射移液管,并顺序将下一个卵母细胞注入管线。
    7. 收集注射的卵母细胞并将其置于装有MBS的6孔组织培养板中。在18℃孵育卵母细胞至少20小时。表达所需的孵育时间取决于正在研究的离子通道。
      注意:对于ENaC的表达,常规注射1-2ng cRNA /亚基,需要24-48小时的孵育。对于其他通道,例如秀丽隐杆线虫退化蛋白通道,注射5-10ng的cRNA /亚单位,并需要更长时间的孵育(> 4天)以获得最佳表达。

  4. 流动装置和层流剪切应力的估计
    1. 利用软件控制的重力流入灌注系统和真空辅助流出灌注用于电生理记录的室(图4)。用于输送流体剪切流的装置由内径为1.8mm的L形玻璃移液管组成,其连接到灌注系统的一个通道。流体剪切移液管由1.8内径毛细管玻璃制成。用本生灯将毛细管玻璃弯曲成L形(〜90°角)。流体剪切移液管安装在记录室前面的三轴手动微操纵器中(见图5B)。


      图4.用于研究流量调节离子通道的记录室和灌注系统的示意图。使用自制(直径为20毫米,深度为6毫米)的室用于洗涤卵母细胞。为了应用层流剪切应力,将自制的垂直灌注玻璃管(1.8mm内径)放置在卵母细胞正上方。流体通过流体剪切移液器通过软件控制的重力灌注系统输送

      图5. Xenopus 卵母细胞的双电极电压(TEV)钳夹。双电极电压钳架的视图。 A.组件的标签如下:1. GeneClamp 500B放大器和Digidata 1550B; 2. VC-6 PTFE阀门灌注系统; 3.具有外部光源的立体显微镜;戴尔OPTIPLEX桌面;左右电极支架,显微操纵器和磁力保持装置;流体剪切移液管,显微操纵器和磁力保持装置; 7.流量阀。 B.仔细观察卵母细胞流动室和灌注系统。

    2. 估计卵母细胞表面上的层流剪切应力(LSS)的速率与拖曳力(F )和卵母细胞表面积的关系LSS = F 拖动 /卵母细胞的表面积。根据F = 0.5ρAω 2 确定F 的大小。 > Cd,其中ρ表示水密度(1g/cm 3),A是球体的横截面积(πr 2 )(对于非洲爪蟾卵母细胞r = 0.05厘米),ω是平均自由流速度(灌注移液管的流速/横截面积),Cd是阻力系数(对于雷诺数在3- 25范围)。雷诺数被确定为R e e =θωD/λ,其中θ是流体密度,ω是平均自由流速度,D是球体直径,λ是水的粘度。例如,使用直径为1.8mm的流体剪切移液管,需要1.5ml/min的灌注速率以产生0.12达因/厘米2的剪切应力。

  5. 双电极电压钳
    1. 实验在室温(20-25℃)下在双电极电压钳架上进行,如图5所示
    2. 估计所需的流速以实现LSS的期望速率,如步骤D2所示。
    3. 灌注注射器与TEV解决方案。将浴液的灌注速率调节至〜3 ml/min。用流量阀将流体剪切移液管的流量调整到所需的速率。流量通过重量测定通过在预加重的管中收集流体一分钟来确定。或者,流量计可以与灌注系统一致地连接以测量流量。
    4. 使用两步协议从微型移液器拉拔器(4台玻璃毛细管(1.5毫米))拉出记录移液器(设定加热器级别步骤1为47.2,步骤2为62.1)。用3 M KCl回填玻璃移液器,轻轻敲打它们,以从尖端释放气泡。
    5. 将注射的卵母细胞转移到记录室的中心(参见图5B)。
    6. 将记录移液器安装在左右电极支架上。放大器处于设置模式时,请小心地将记录移液器置于腔室中,以测量TEV溶液中的电阻。
      注意:移液器的电阻应在0.2-2mΩ之间。具有电阻的移液器& 0.1mΩ被认为是泄漏的,应该更换。

    7. 将记录电极的偏移调整为零。
    8. 通过改变显微操纵器的X-Y-Z旋钮将记录电极移动到靠近卵母细胞的位置。使用Z旋钮轻轻地将记录电极刺入卵母细胞。
    9. 液体剪切移液管应使用立体显微镜可见。使用立体显微镜,将流体剪切移液管的尖端直接置于卵母细胞上方。在卵母细胞顶部0.5-1.0毫米内小心移动流体剪切移液管。
    10. 将放大器设置为电压钳模式。
    11. 对于ENaC,电流以-60 mV的膜电位连续记录。灌注系统使用pClamp 10通过外部TTL信号进行控制。在记录过程中,腔室连续灌注TEV溶液。通过流体剪切移液管施加流体脉冲。通常,我们在头30秒内用电泳灌注记录电流,我们应用流体剪切应力60秒或直到当前平台,然后用含有苯甲酰胺(5μM)的TEV溶液灌注室30秒以确定泄漏电流。
    12. 为了检查流动激活组件的电压依赖性,在基础条件下,使用一系列电压步骤(例如 500毫秒)测量全电池电流,从-140至60mV,在流体剪切应力的存在和用通道抑制剂灌注后(详见2006年)(参见Carattino等人,2006)。
    13. 在实验结束时,将流体剪切移液管移动到初始位置,从卵母细胞中取出记录移液器,弃掉卵母细胞。
      注意:在实验过程中使用的苯甲酰胺或其他通道阻滞剂必须通过在浴液和垂直管中冲洗TEV溶液至少3分钟来洗掉。

数据分析

  1. 对于ENaC和MEC信道,对LSS(LSS响应)的响应的大小被定义为在存在LSS的情况下电流的苯甲酰敏感敏感成分(Iα> LSS I benz )来确定I-subSIS 的浴液中的苯甲酰胺,与在LSS(基础 )开始之前测量的当前值相当。通过将不敏感的电流减去由LSS( 引发的峰值电流来计算I + LSS >)。


    图6.流程介导的C激活。线虫在非洲爪蟾卵母细胞中的MEC-4/MEC-10通道
    。向卵母细胞注射编码MEC-4/MEC-10的cRNA。 LSS(0.12达因/厘米2))通过液体剪切移液管连续浴灌注。膜电位钳位在-60mV。在t = 30秒时开始垂直流量为1.5ml/min(灰条)。在实验结束时,通过浴(黑条)灌注含有苯甲酰胺(5μM)的TEV溶液。进行了三次测量:基线电流(基线 ),LSS刺激电流( I )和苯甲酰不敏感的电流( I )。 MEC-4/MEC-10信道对LSS(LSS响应)的响应的大小被估计为I + LSS /sub>。

  2. 通道激活的时间常数(τ)通过将垂直灌注开始后的前60秒电流增加拟合到指数方程来确定:

    >
    其中,
    I 是宏观电流,
    是时间常数,
    a c 是曲线拟合确定的常数。

笔记

由于卵母细胞质量的季节变化,胶原酶处理程度和cRNA注射程序,卵母细胞中蛋白质的表达存在固有的变异性。为了说明表达的变异性,用从不同青蛙收获的几批卵母细胞重复实验。

食谱

  1. Ca 2 +无标准卵母细胞溶液(SOS),RT
    100 mM NaCl
    2mM KCl
    5 mM HEPES
    使用1N NaOH调节pH至7.4 存放于室温(室温)
  2. 改良巴斯溶液(MBS)
    88 mM NaCl
    1 mM KCl
    24mM NaHCO 3
    15 mM HEPES
    0.3mM Ca(NO 3 3)2
    0.41mM CaCl 2
    0.82mM MgSO 4
    10μg/ml青霉素钠
    10μg/ml硫酸链霉素
    100μg/ml硫酸庆大霉素
    使用1N NaOH调节pH至7.4 储存于18°C
  3. 低调解决方案
    100mM K 2 HPO 4
    1克/升BSA
    将pH调节至6.5
    存储在RT
  4. 双电极电压钳记录解决方案(TEV)
    110 mM NaCl
    2 mM KCl
    1.54mM CaCl 2
    10 mM HEPES
    使用1N NaOH调节pH至7.4 存储在RT
  5. Benzamil工作解决方案
    在DMSO中制备0.1M苯甲酰胺储存液,并保持黑暗
    在实验当天新鲜稀释至终浓度为TEV溶液(见配方4)

致谢

这项工作得到了NIH授权R01DK084060(M.D.C.),K01DK103834(S.S.)和匹兹堡肾脏研究中心的细胞生理学和肾脏成像核心(P30-DK079307)的支持。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Shi, S. and Carattino, M. D. (2017). Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes. Bio-protocol 7(8): e2224. DOI: 10.21769/BioProtoc.2224.
  2. Shi, S., Luke, C. J., Miedel, M. T., Silverman, G. A. and Kleyman, T. R. (2016). Activation of the Caenorhabditis elegans degenerin channel by shear stress requires the MEC-10 subunit. J Biol Chem 291(27): 14012-14022.
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