CHAPTER

Reconstitution of lysosomal ion channels into artificial membranes

11

Elisa Venturi, Rebecca Sitsapesan1 Department of Pharmacology, University of Oxford, Oxford, UK 1

Corresponding author: E-mail: [email protected]

CHAPTER OUTLINE Introduction ............................................................................................................ 218 1. The Bilayer Apparatus ...................................................................................... 218 2. Electrical Equipment Used for Single-Channel Recordings .................................. 220 3. Painting Bilayers.............................................................................................. 222 4. Ion Channel Incorporation into a Bilayer............................................................ 223 4.1 Fusion of Native Vesicles or Purified Proteins with the Bilayer ............. 223 4.2 Ion Channel Orientation ................................................................... 224 5. Single-Channel Current Amplitude and Conductance Measurements .................... 225 6. Choice of Permeant Ion .................................................................................... 227 6.1 Native Ion Channels ........................................................................ 227 6.2 Recombinantly Expressed and Purified Ion Channels .......................... 227 7. Measuring the Relative Permeability of Different Ions......................................... 229 8. Measurements of Liquid Junction Potentials....................................................... 230 9. Single-Channel Gating and Measurements of Open Probability ............................ 230 10. Noise Analysis ................................................................................................. 231 11. Isolation of Native and Recombinant Purified Lysosomal Ion Channels................. 232 11.1 Native Lysosomal Ion Channels ........................................................ 232 11.2 Purification of Recombinantly Expressed Lysosomal Channels ............. 233 11.2.1 Purification of human TPC1 overexpressed in HEK293 cells ........ 233 12. Discussion ....................................................................................................... 234 References ............................................................................................................. 234

Abstract Ion channels that are located on intracellular organelles have always posed challenges for biophysicists seeking to measure their ion conduction, selectivity, and gating kinetics. Unlike cell surface ion channels, intracellular ion channels cannot be accessed for Methods in Cell Biology, Volume 126, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2014.10.023 © 2015 Elsevier Inc. All rights reserved.

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biophysical single-channel recordings using the patch-clamp technique while remaining in a physiological setting. Disruption of the cell is always necessary and hence experiments inevitably have a certain “artificial” nature about them. This drawback is turned to considerable advantage if the internal membranes containing the channels of interest can be isolated or if the channels can be purified because they can then be incorporated into artificial membranes of controlled composition. This approach guarantees a tight but flexible control over the biophysical and biochemical environment of the ion channel molecules. This includes the lipid composition of the membrane and the ionic solutions on both sides of the channel, thus allowing the conductance properties of the channel to be accurately measured. Since the influence of multiple unknown regulators of channel function (that could be present within the physiological membrane or in cytosolic, or intraorganelle compartments) is removed, the identification and characterization of physiological and pharmacological regulators that directly affect channel gating can also be achieved. This cannot be performed in a cellular environment. These techniques have typically been used to study the properties of channels located on endoplasmic/sarcoplasmic reticulum (ER/SR) membranes but in this chapter we describe how the techniques are also suited for ion channels of the acidic lysosomal and endolysosomal Ca2þ stores.

INTRODUCTION This report will focus on the techniques of producing artificial planar phospholipid bilayers, the incorporation of lysosomal and endolysosomal ion channels into those membranes, and the recording and analysis of the subsequent single-channel current fluctuations that are obtained under voltage-clamp conditions. Of course, before such experiments can be undertaken, it is essential to prepare an enriched membrane preparation containing the ion channel of interest either in native membrane vesicles or reconstituted proteoliposomes. These methods are described in brief in section Isolation of native and recombinant purified lysosomal ion channels as they are under constant revision and refinement. We show examples of the recordings of nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive ion channels from both native lysosomal membranes and from a purified preparation of two-pore channel type 1 (TPC1) proteins (section Purification of human TPC1 overexpressed in HEK293 cells). There is no single “best” method for reconstituting ion channels into planar phospholipid membranes but we here describe effective protocols that are used in our laboratory.

1. THE BILAYER APPARATUS The protocol presented here is adapted from the method first described by Miller (1978). The presence of charged lipids within a membrane can affect the function of ion channels and complicate interpretation of biophysical data. For this reason, it is beneficial to begin the characterization of a novel ion channel using the most simple, uncharged membrane composition. We therefore paint bilayers using

1. The bilayer apparatus

phosphatidylethanolamide (PE) purchased from Avanti Polar Lipids. The purified PE lipids in powder form are dissolved in chloroform at a concentration of 50 mg/mL and stored at 80  C in glass vials. Prior to use, the chloroform is evaporated off under a stream of nitrogen gas and the lipids are resuspended in decane to obtain a final concentration of 35 mg/mL. Solvent resistant tips are used to minimize contamination of the lipid stocks. The bilayer apparatus includes a block and a Delrin cup (acetyl resin) (Warner Instruments) as illustrated in Figure 1. The cup has an aperture of 150 mm diameter. The block has a figure 8-shaped cavity in which the cup can be accommodated creating two distinct compartments (cis and trans), which are connected by the aperture. Two smaller holes on one side of the block are filled with 3 M LiCl solution. These cavities accommodate both Ag/AgCl electrodes and agar bridges. The agar bridges function as an electrical conduction pathway between the LiCl solution and the experimental recording solutions contained in the cis and the trans chambers. The agar bridges are made of U-shaped borosilicate capillaries filled with a

FIGURE 1 Bilayer recording apparatus. Diagram of the block and cup illustrating the design of the chambers and the dimensions.

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solution containing 3 M LiCl and 2% agar. An illustration of the electrical connections between electrodes and the compartments obtained via the agar bridges is shown in Figure 2.

2. ELECTRICAL EQUIPMENT USED FOR SINGLE-CHANNEL RECORDINGS The electrical equipment that we use to acquire single-channel recordings is currently comprised of the following: •



• • •

BC-525D Bilayer Clamp Amplifier (Warner Instruments) and a capacitive feedback headstage to voltage-clamp the bilayer and record current fluctuations across the membrane. Digidata 1440A (Axon Instruments), a data acquisition unit that is connected to a computer using a dedicated host interface: it allows the conversion of the analogue signal from the amplifier into a digital form (analogue/digital (A/D) converter). A computer for data acquisition and storage. A low-pass 8 pole Bessel filter (Frequency Devices Inc) to filter the signal at an appropriate frequency (600e1000 Hz) before displaying it on the oscilloscope. Oscilloscope (Hitachi VC-6545) to display in real time the data as it is recorded.

FIGURE 2 Diagram illustrating the block and cup and the electrical pathways between electrodes and the two compartments. The bilayer formed across the 150 mm aperture in the cup, separates the cis and trans chambers. The electrodes and the agar bridges are placed in the small LiCl chambers so that the electrodes and the experimental solutions contained in the cis and trans chambers are electrically coupled. The voltage input is commanded by the headstage which holds the cis chamber at potentials relative to ground (trans chamber).

2. Electrical equipment used for single-channel recordings





A magnetic stirring system (Spin-2 stirplate, Warner Instruments) on which the block and the cup are placed. This permits mixing of the solutions in the chambers using small magnetic stir bars in the chambers. A Faraday cage for shielding of electrical noise in which the bilayer support, headstage, and stirplate are accommodated.

Achieving high signal-to-noise ratios in single-channel recordings is essential for accurately determining the current amplitudes and temporal resolution of ion channel gating transitions. While the signal-to-noise ratio is intrinsically limited by factors such as background noise, parasitic capacitances of the acquisition electronics and the lipid membrane, great care must be taken in circuit grounding to avoid ground loops. In Figure 3, a diagram of the electrical equipment and an example of a circuit grounding scheme are shown. In this configuration, the central grounding point of the system is the CIRCUIT GROUND located on the rear panel of the BC525D amplifier. The stirplate is connected to the Faraday cage, which is grounded directly to the central ground point on the amplifier. The headstage ground coincides with the amplifier ground. For real-time monitoring of the current fluctuations arising from the openings of a channel incorporated into the bilayer, the analogue signal from the amplifier can be low-pass filtered and displayed on an oscilloscope. The level of filtering selected for visualization, usually around 600e800 Hz, must be adequate for removing most

FIGURE 3 Electrical equipment and example of a circuit grounding configuration. The headstage and the bilayer chambers apparatus are protected from electrical background noise by the insulating properties of the Faraday cage. The stirplate is directly grounded to the Faraday cage, which is earthed to the main circuit ground on the BC-525D amplifier. Any current flowing across the bilayer (through an open ion channel) is acquired by the amplifier and forwarded to both a low-pass 8 pole Bessel filter for visualization on the oscilloscope and the A/D converter interface for recording/storage of the signal onto the computer.

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of the background noise and allowing visualization of the single channel openings. The signal output is also simultaneously digitalized by an A/D converter and stored on computer. The sampling rate of the digitization should be carefully selected to avoid distortion (aliasing) of the signal. According to the NyquisteShannon sampling theorem (Eqn (1)), the signal can be accurately reconstructed, without loss of information and aliasing, if the sampling rate, fs, is at least twice the component with the highest frequency, B, contained in the signal (Nyquist, 1928; Shannon, 1948). fs > 2B

(1)

For example, if the signal output from the amplifier is low-pass filtered at 10 kHz, the highest frequency of the recording corresponds to half of the cut-off frequency of the low-pass filter (5 kHz) (Colquhoun & Sigworth, 1983). A conservative sampling interval set between 5 and 10 times this value, would be sufficient for an accurate signal reconstruction (25e50 kHz). After digitization, the stored single-channel recordings should be further lowpass filtered for practical analysis of the channel gating and conductance properties. Low-pass filtering of electrophysiological records is required because of the presence of the intrinsic background noise of the recording devices and the interferences from electrical mains (50 Hz). The higher frequencies contained in the background noise can be eliminated by a low-pass filter without any distortion of the original signal. In case of recordings where the open channel current amplitudes are very small and, therefore, the signal-to-noise ratio is low, a typical level of filtering is 600e800 Hz. It is important to realize that the higher the degree of filtering, the lower the time resolution of the single-channel traces and the capacity for detecting brief events. For a detailed analysis of the data acquisition and filtering levels to use for the correct examination of a single-channel record, see Colquhoun and Sigworth, (1983).

3. PAINTING BILAYERS The Delrin cup forms the bilayer support structure and it is across its 150 mm aperture that the artificial membrane is formed or “painted.” The cup is usually stored in a diluted detergent solution (Fairy Liquid, Procter and Gamble) and rinsed thoroughly before use. The first crucial step for forming a stable bilayer, suitable for reconstitution experiments, lies in the “priming” of the bilayer aperture. This “priming” procedure consists in depositing a small drop of lipid suspension (approximately 0.1 mL) onto the aperture using a 10 mL pipette tip. The cup is left to dry for at least 20 min and then placed in the block to form the two partitions. The cis and trans chambers are then filled with the same electrolytic solution to create symmetrical ionic conditions. These solutions will have different ionic composition according to the ion channel under investigation and the experiment to be carried out (see below for Choice of permeant ion). For bilayer formation, a small amount of PE

4. Ion channel incorporation into a bilayer

lipids is “painted” across the hole from the inside of the cup (cis chamber) using a painting stick fashioned from a plastic transfer pipette with a tip of approximately 1 mm. The first application of lipids forms a layer of several microns in thickness surrounded by an annulus or torus of lipids in direct contact with the perimeter of the hole in the cup (Tien, 1968). This film can thin spontaneously to a planar bilayer due to the combined effects of the curvature of the torus and the Londonevan der Waals forces generated between the two aqueous phases on opposite sides of the bilayer (White, 1972). Bilayer formation is readily monitored by measuring the membrane capacitance using the capacitance test implemented in the BC-525D amplifier. Briefly, the amplifier commands a triangular waveform to the bilayer system while monitoring the amplitude of the resulting wave. If a bilayer is not formed across the aperture, the amplifier output will be the same 10 V peak to peak triangular signal applied. The formation of a bilayer introduces a capacitor into the circuit and this results in a square waveform with amplitude proportional to the membrane capacitance (“Bilayer Clamp Amplifier Manual Model BC-535,” Retrieved August 4, 2014). Using our experimental system, PE bilayers formed across a 150 mm aperture of a Delrin cup that are suitable for ion channel incorporation and single-channel recordings, exhibit a capacitance in the range 80e140 pF.

4. ION CHANNEL INCORPORATION INTO A BILAYER 4.1 FUSION OF NATIVE VESICLES OR PURIFIED PROTEINS WITH THE BILAYER After a stable bilayer is obtained, ion channel reconstitution can be achieved with the formation of a gradient of salt across the membrane. This is generated by addition to the cis chamber of 100e200 mL of a high-salt solution (3 M). This osmotic gradient allows the swelling and the subsequent fusion of native vesicles or proteoliposomes when they are added to the cis compartment and continuously stirred. Vesicles and proteoliposomes first move toward the osmotic gradient (cis to trans) before they finally adhere to the membrane. Movement of water across the bilayer in the trans to cis direction causes enlargement of the adherent vesicles until these burst and fuse with the membrane (Akabas, Cohen, & Finkelstein, 1984; Cohen, Zimmerberg, & Finkelstein, 1980). A diagram of the osmotic gradient and the trans-bilayer water flow that occurs under these conditions is illustrated in Figure 4. Ion channel insertion into the bilayer can be easily assessed by continuously monitoring the current flowing across the bilayer. Sudden step increases in current are observed upon a fusion event. After ion channel incorporation, the cis and the trans solutions are replaced with appropriate recording solutions according to the required experimental protocol and the particular ion channel of interest. Perfusion of the cis chamber first is recommended in order to avoid further vesicle/proteoliposome fusion events. The perfusion can be obtained using either a peristaltic pump

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FIGURE 4 Effects of the application of an osmotic gradient across the bilayer. After an osmotic gradient between the ionic solutions in the cis and trans chamber is created, water molecules begin to move across the membrane in the area of contact with the vesicle/ liposome membrane, entering the vesicles/proteoliposomes and producing swelling (Akabas et al., 1984; Cohen et al., 1980).

with an inflow and outflow system or a gravityefeed system composed of a reservoir and a syringe for aspiration. Exchange of the solution will be assured when an inflow of approximately 10 times the volume of the compartment (10 mL) is perfused into the chamber. Once the chambers are perfused with the desired recording solutions, the bilayer is subsequently held at different holding potentials in order to obtain single-channel currentevoltage relationships, examine the selectivity and permeability properties of the ion channel of interest or study channel gating behavior as required.

4.2 ION CHANNEL ORIENTATION It is very important to ascertain the orientation of an ion channel once it has inserted into a bilayer since membrane vesicles and proteoliposomes may contain ion channels of mixed orientation. Channel orientation after incorporation into a bilayer can be tested in various ways depending on the characteristics of the particular ion channel of interest. For example, changes in holding potential can identify orientation for voltage-dependent channels, or the use of membrane-impermeant channel pore blockers or specific ligands, which bind only to one side of the channel, can also be applied. Using the techniques described above, certain channels always incorporate into the bilayer in a fixed orientation; the ryanodine receptor (RyR) is a prime example (Sitsapesan & Williams, 1994). TPC1 and the two-pore channel type 2 (TPC2) appear to incorporate such that the cytosolic side of the channels face into the cis chamber, as judged by the voltage-dependence or use of the channel modulators, NAADP, Ned-19, pH and Ca2þ (Pitt et al., 2010; Pitt, Lam, Rietdorf, Galione, & Sitsapesan, 2014). However, changes to the methods used to isolate lysosomal membrane vesicles or purify the channels may alter the orientation of the channels in the bilayer.

5. Single-channel current amplitude and conductance measurements

5. SINGLE-CHANNEL CURRENT AMPLITUDE AND CONDUCTANCE MEASUREMENTS Ion channels are pore-forming proteins that provide a conductive pathway across the dielectric barrier formed by the lipid membrane. The permeability and ionic selectivity properties of a channel will determine which ions can move through the channel pore. “Permeability” refers to the ability of an ionic species to diffuse through the channel, while “selectivity” is the ability of the channel to discriminate between different ions. The single-channel conductance (measured in siemens (S)) for a given ion is the rate of ions that travel through a single open channel and depends on the permeability and selectivity of the channel. An ion channel inserted into the bilayer can be represented as a conductor and thus, from Ohm’s law: I ¼ gV

(2)

where I is the current flowing through the conductor, g is the conductance and V is the potential difference (membrane potential) across the conductor. Obtaining measurements of current amplitude for a range of holding potentials for a given ion channel in a known ionic condition, allows the construction of a currentevoltage relationship or I/V plot. The unitary conductance of the channel under investigation can then easily be obtained using linear regression interpolation of the points. An example of such a plot is shown in Figure 5 for TPC1 in symmetrical 210 mM Kþ solutions. Current amplitudes can be directly measured from the single-channel recordings using manually controlled cursors in most commercial single-channel software. An alternative method for measuring current amplitudes (which is not always suitable for channels which only open rarely or briefly to the full open state) is provided by the use of the so-called all-points amplitude histogram. This method is based on the construction of a histogram from all the digitized points in the recorded singlechannel trace. The amplitude histogram of a single-channel gating between open and closed states will have two peaks, one corresponding to the zero level current (closed) and the other to the open channel level. The resulting histogram is then usually fitted with a Gaussian curve with the appropriate number of exponential terms (one for each peak). The fitting curve follows the formula of the Gaussian distribution f(x): 2 2   Xn eðxmi Þ =2si p ffiffiffiffiffi ffi f x ¼ A i¼1 i si 2p

(3)

where x is the single-channel current amplitude; i is the number of Gaussian curves; Ai, mi, and si are the area; the mean and the standard deviation of the ith Gaussian component, respectively. The detection of current amplitudes using the all-points amplitude histogram method is particularly useful for the identification of subconductance states within a single-channel recording. A subconductance state is one in which the amplitude of the open event is lower than the fully open channel level. Subconductance gating

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FIGURE 5 A representative example of a single TPC1 channel gating in the bilayer with KD as permeant ion and corresponding currentevoltage relationship. Kþ current fluctuations through a purified TPC1 channel reconstituted into a bilayer in symmetrical 210 mM KCl solutions at holding potentials of þ40 mV, þ60 mV, þ80 mV, and 60 mV as shown. Note that at positive potentials, channel opening events are upward deflections (Kþ ions flowing in the cis to trans direction) and at negative potentials are downward deflections (Kþ ions flowing in the trans to cis direction). O and C indicate the open and closed channel levels, respectively. The recording was low-pass filtered at 800 Hz. The corresponding TPC1 currentevoltage relationship obtained from the same experiment, gives rise to a conductance value of 83 pS. The TPC1 channels used in this experiment were purified from HEK293 cells overexpressing human TPC1 as described in section Purification of human TPC1 overexpressed in HEK293 cells.

states can be induced by the binding of agents within the channel pore to impede the flux of permeant ion (called a “blocker”) or by agents/modulators or mutations that produce conformational changes to the pore region. The accuracy of estimating full conductance or subconductance state amplitudes can be limited by the following factors: 1. High background noise so that the recording is characterized by a poor signal-tonoise ratio and the histogram does not effectively distinguish between fully open and subconductance states. 2. The channel Po is very low and the opening events are too few and too brief to fully resolve. 3. The channel may exhibit a “noisy” open level. This increases the variation in the distribution making it more difficult to identify the open, closed, and subconductance levels.

6. Choice of permeant ion

4. Baseline drift can mask the appearance of subconductance gating states and cause poor resolution of the peaks.

6. CHOICE OF PERMEANT ION 6.1 NATIVE ION CHANNELS Membrane vesicles from lysosomes or endolysosmes can be fused with artificial membranes to investigate the functional properties of the “native” ion channels in those membranes. While single-channel function might be better preserved under native conditions (because detergents are not necessary and fewer potentially damaging steps such as freeze/thawing or warming of the proteins are undertaken), certain restrictions in the ionic composition of the experimental solutions will be required. For example, in order to observe cation currents through native TPC channels, Cl must be replaced by anions that are impermeant in the Cl channels that are present in those membranes (for example, piperazine-1,4-bis-2-ethanesulfonic acid (PIPES) or methanesulfonate) to eliminate possible interference of anion currents as observed in whole lysosome patch-clamp studies (Schieder, Ro¨tzer, Bru¨ggemann, Biel, & Wahl-Schott, 2010a; Schieder, Ro¨tzer, Bru¨ggemann, Biel, & Wahl-Schott, 2010b). The same principle must be applied when using Ca2þ as the permeant ion. An example of recording solutions used in experiments where the Ca2þ permeability properties of TPC2 are under investigation is described below: • •

cis chamber: 250 mM HEPES, 125 mM Tris, pH 7.2, free [Ca2þ] 10 mM but this can be adjusted by additions of EGTA and CaCl2 trans chamber: 250 mM glutamic acid, 10 mM HEPES and brought to pH 7.2 with Ca(OH)2 (free [Ca2þ] w50 mM)

Under these ionic conditions, Ca2þ ions fluxes in the trans to cis direction can be recorded when a lysosomal/endolysosomal Ca2þ permeable channel incorporates into the bilayer. These solutions have been extensively used in the single-channel studies of native RyR and, therefore, allow the direct comparison of the conductance and permeability properties of these two distinct families of intracellular Ca2þ release channels. An example of a native Ca2þ permeable channel recorded from isolated lysosomal vesicles and reconstituted into a bilayer is shown in Figure 6.

6.2 RECOMBINANTLY EXPRESSED AND PURIFIED ION CHANNELS When using a purified ion channel preparation, since no other ion channel will incorporate into the bilayer alongside the ion channel under investigation, there are no restrictions to the ionic composition of the solutions either side of the bilayer. This enables a comprehensive investigation of the conductance properties of the channel. A typical example of the single-channel current fluctuations and

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FIGURE 6 Typical example of Ca2D current fluctuations obtained after fusion of lysosomal vesicles with a planar lipid bilayer. Membrane vesicles were prepared from HEK 293 cells overexpressing human TPC2 (as described in section Native lysosomal ion channels) and were fused with a PE bilayer. The single-channel openings were obtained in asymmetrical Ca2þ solutions (Tris/HEPES, 10 mM free Ca2þ, pH 7.2 cis /50 mM Ca2þ glutamate, pH 7.2 trans) at a holding potential of -60 mV. This potential was chosen because of the expected small Ca2þ conductance of TPC2 under these conditions (15 pS). With these recording solutions, Ca2þ is the only permeant ion and therefore only the opening of Ca2þ -permeable ion channels will be observed if vesicles containing these channels fuse with the bilayer. When the channels open, current will flow across the bilayer in the trans to cis direction, and will be seen as upward deflections as shown in the illustrated traces. O and C indicate the open and closed channel levels, respectively. The record was low-pass filtered at 600 Hz. In this experiment, only a few very brief opening events were observed under control conditions (top trace). Addition of nanomolar NAADP on the cis side (which probably corresponds to the cytosolic side) of the channel increased the number of channel openings. Subsequent addition of nanomolar Ned19 potentiates the effects of NAADP. 1 mM Ned-19 subsequently completely inhibited channel openings. These effects of NAADP and Ned-19 on the native lysosomal cation channel are similar to the effects that we observe in our laboratory when human purified TPC2 channels are reconstituted into the bilayer (for example, (S. J. Pitt et al., 2010)).

corresponding currentevoltage relationship of a human recombinant, purified TPC1 channel recorded in our laboratory is shown in Figure 5. The recording was obtained in symmetrical (both cis and trans) solutions of 210 mM KCl, 10 mM HEPES at pH 7.2. If the purified ion channel properties are unknown, the first important step is to identify whether it is more selective for cations over anions or vice versa. This is generally achieved using a gradient of KCl between the cis and trans chambers. Under these conditions, the reversal potential (Erev), which is the potential at which the

7. Measuring the relative permeability of different ions

net transmembrane flux is zero, can be measured and compared with the predicted reversal potential for Kþ from the Nernst equation which is as follows (Hille, 1992):  þ K RT ln þ out Erev ¼ (4) zF ½K in where R, the ideal gas constant, T, the temperature in kelvins, F is Faraday’s constant, z is the valence of the Kþ ions (þ1), and [Kþ]out and [Kþ]in are the Kþ concentrations in the trans and cis compartments, respectively. The calculation can be repeated to find the Erev for Cl ions. For example, if the Erev measured from the currentevoltage relationship obtained under gradient conditions is close to the calculated reversal potential for Kþ, this indicates that the ion channel is selective for cations over anions.

7. MEASURING THE RELATIVE PERMEABILITY OF DIFFERENT IONS Ion permeability and selectivity of biological membranes can be described by the GoldmaneHodgkineKatz (GHK) electrodiffusion theory (Hodgkin & Katz, 1949) from which the GHK voltage equation is derived:     ! P P PMiþ Miþ out þ PNj Nj in RT P ln P Erev ¼ (5) F PMiþ ½Miþ in þ PNj ½Nj out In this format, the equation assumes that there are Mi monovalent positive ionic species and Nj negative ionic species in the solutions on either side of the membrane. Erev is the reversal potential or zero-current potential, which is the potential at which there is no current flow. P is the permeability for that ion, R, the ideal gas constant, T, the temperature in kelvins, and F is Faraday’s constant. From this equation, if the ion concentrations are known, it is possible to calculate the relative permeability ratios for different ionic species by measuring the reversal potential. In the presence of one permeant ion on either side of the membrane (for example, ion A on one side and ion B on the other side) and with the same valence z, the simple biionic equation can be derived as follows: Erev ¼

RT PA ½Ao ln zF PB ½Bi

(6)

When the ions have different valences such as Ca2þ and Kþ, the permeability ratio for divalent over monovalent ions can be obtained using the Fatt and Ginsborg’s equation (1958) (Fatt & Ginsborg, 1958) as:             PX 2þ PY þ ¼ Y þ 4 X 2þ exp Erev F RT exp Erev F RT þ 1 (7) where Erev, R, T, and F have their usual meanings. This equation is used, for example, for the calculation of the PCa2þ/PKþ. The Erev obtained from a

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currentevoltage relationship must be first corrected for the liquid junction potentials (LJPs) arising between the different solutions (see below).

8. MEASUREMENTS OF LIQUID JUNCTION POTENTIALS When two electrolytic solutions of different concentration and ionic composition are in contact, an LJP arises at the interface between the solutions (Barry & Lynch, 1991). This is because of differences in the properties of the ions between the two solutions, in particular, their mobility, and concentration in each solution (Bockris & Reddy, 1970). The ionic mobility is the ability of an ion to move through a medium in response to an electric field and it is dependent on size, charge, hydration, and temperature (Bockris & Reddy, 1970). During bilayer experiments, the two bathing solutions contained in the cis and trans chambers may set up an LJP which will cause an offset when the bilayer is voltage-clamped. The LJPs can be ignored when measuring the conductance of an ion channel; the IeV relationship will be shifted by a few millivolts but the slope of the curve will be retained. On the other hand, when the permeability properties of an ion channel are under investigation, it is mandatory to obtain an accurate reading of the voltage. In this case, corrections for the LJP must be applied (see section above). The magnitude and the direction of the LJP can be estimated using Clampex 10.3 (Axon Instruments), which integrates an adaptation of the software developed by Barry (1994) (JPCalc).

9. SINGLE-CHANNEL GATING AND MEASUREMENTS OF OPEN PROBABILITY An ion channel is a single molecule in continuous rapid transition between two main conformational states; an open state in which ions can permeate through the channel pore and a closed state in which the channel does not conduct ions. The opening and closing of an ion channel is termed “gating.” The opening event of a single channel incorporated in a bilayer, produces a current step in amplitude in the order of picoAmperes (pA), and the closing event occurs when the current amplitude returns to zero. The analysis of the time spent by the channel in each state (dwell times) provides information about the single-channel function. The gating parameters obtained can then be compared under different experimental conditions in order to examine, for example, whether a ligand affects the likelihood of finding the channel open. In other words, whether the ligand affects the open probability, (Po) of the channel. Analysis of the gating of an ion channel is generally carried out using commercial software and may be complicated by factors such as multiple conducting states (often referred to as subconductance open states), the simultaneous gating of multiple channels present in the bilayer or a very low signal-tonoise ratio. In the latter condition, the software is most likely to detect false events and generate errors in computing the gating parameters of the channel.

10. Noise analysis

Detection of the opening and closing channel transitions can be achieved using various methods including threshold-based methods (Colquhoun & Sigworth, 1983), time-course fitting (Colquhoun & Sigworth, 1983), and more complicated algorithms based on hidden Markov models (for example, Qin, 2004). The most common method used is the 50% threshold method (Colquhoun & Sigworth, 1983). This method is mainly used for ion channels which gate between two, well-defined closed and open states and is routinely applied because it is implemented in many of the commercially available software. Every time the amplitude of the current crosses the 50% threshold level (50% of the single channel open state amplitude) the transition is detected and recorded as an opening or closing event of the channel. Each state transition and duration can be recorded and saved in an event file, which can subsequently be analyzed for interpreting the ion channel behavior. The most significant statistical parameter is the open probability (Po) of the single channel. Equation (8) shows how the Po is computed by the software: Po ¼

Topen Topen þ Tclosed

(8)

where Topen and Tclosed are the times spent in each state, respectively. When multiple channels are reconstituted into the membrane patch, the NPo is determined by the following formula:       Topen1 þ 2 Topen2 þ 3 Topen3 þ .n Topenn NPo ¼ (9) Ttotal where N is the number of channels gating in the bilayer, Topen1, Topen2, and Topen3 are the duration times in which the channels open in the first, second, and third level, respectively and Ttotal is the total time of the recording. From Eqn (9), knowing the number of channels, N, it is possible to calculate the average Po (avgPo) of the channels gating in the bilayer: NPo Po1 þ 2Po2 þ 3Po3 þ .nPon ¼ (10) N N where Po1, Po2, and Po3 are the probability of dwelling in the first, second, or third channel level, respectively. avgPo ¼

10. NOISE ANALYSIS Noise analysis can be used to obtain information about ion channel behavior even when multiple channels have incorporated into the bilayer. The channels reconstituted in the membrane randomly open and close and the sum of all the channel fluctuations creates a mean current or a “noise” current. This time-averaged current, resulting from a large number of channels simultaneously gating, is computed using simple algorithms, which are embedded in various single channel software, for example, in WinEDR (John Dempster, Strathclyde University, UK). In general,

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the current fluctuations across the bilayer are subdivided into multiple segments in time with each segment containing N samples. The mean current for each segment can be calculated with the formula, PN IðiÞ (11) Imean ¼ i¼1 N where I(i) is the amplitude of the ith current of the N samples in the segment. The mean current values can then be plotted against time. The analysis of the mean current and its variation is considered a useful tool for detecting changes in the ion channel gating behavior after an intervention such as the addition of a modulator or a change in the voltage. It is important to consider that this kind of analysis is normally carried out with the assumption that there is no variability in gating behavior within the ion channel population.

11. ISOLATION OF NATIVE AND RECOMBINANT PURIFIED LYSOSOMAL ION CHANNELS 11.1 NATIVE LYSOSOMAL ION CHANNELS Several methods have been developed for isolating lysosomes. These include fractionation methods, based on size and density of the subcellular compartments (Kawashima et al., 1998; Storrie & Madden, 1990; Zhang & Li, 2007), differential centrifugation methods (Schenkman & Cinti, 1978), and magnetic chromatography procedures (Diettrich, Mills, Johnson, Hasilik, & Winchester, 1998; Duvvuri & Krise, 2005). Here we describe the methods (based on those described by Duvurri and Krise (2005) that were adapted from the procedure originally developed by Diettrich et al. (1998)) that we used to isolate lysosomes from HEK293 cells stably overexpressing TPC2. The membranes were then fused with artificial membranes to examine the biophysical properties of native TPC2 (and any other ion channels present in the lysosomal membranes). An example of the ion channels observed from this lysosomal preparation is shown in Figure 6. HEK293 cells stably overexpressing His-tagged human TPC2 are incubated with Feedextran particles at a concentration 2 mg/mL for 1 h at 37 C in culture medium to allow endocytic uptake. Cells are then carefully washed 4 times with PBS and subsequently cultured for 24 h in Feedextran-free culture medium to allow accumulation of Feedextran in the lysosomal lumen. Cells were first washed with PBS and then homogenized in a Dounce homogenizer in a hypotonic buffer of the following composition: 15 mM KCl, 1.5 mM Mg(OAc)2, 1 mM DTT, and 10 mM HEPES, pH 7.4 supplemented with a protease inhibitor cocktail ((Roche Applied Sciences) and 0.5 mg/mL DNase I). Isotonic conditions were reestablished by the addition of 375 mM KCl, 22.5 mM Mg(OAc)2, 1 mM DTT, and 10 mM HEPES, pH 7.4, and 20% (v/v) of the homogenate volume. The suspension is centrifuged at 700  g for 10 min and the resulting postnuclear supernatant is passed through a midiMacs

11. Isolation of native and recombinant purified lysosomal ion channels

LS column (Miltenyi Biotec, US) contained in a magnetic sleeve. Nonspecific binding of organelles to the column is achieved via preequilibration with 500 mL of 0.5% bovine serum albumin in PBS. The midiMacs column is then washed twice with icecold PBS containing 0.5% bovine serum albumin and 0.5 mg/mL DNase I. Ten minutes of incubation in the presence of 0.5 mg/mL of DNase I then facilitates the break up of organellar aggregates that may have formed during homogenization. The column is washed with two more volumes of PBS with 0.5% bovine serum albumin. Elution of the purified lysosomes via gravity flow is obtained by adding 1 mL of PBS after the column is removed from the magnetic sleeve. The isolated lysosomes are then centrifuged at 15,000  g for 10 min and resuspended in a buffer containing 50 mM KCl, 10 mM NaCl, 60 mM KF, 20 mM EGTA, 10 mM HEPES, pH 7.2.

11.2 PURIFICATION OF RECOMBINANTLY EXPRESSED LYSOSOMAL CHANNELS Different ion channels require different purification procedures to ensure that sufficient pure protein is obtained for the required experiments and that the functional properties of the ion channel are retained. Below is the purification procedure that we used to functionally purify TPC1. His-tagged TPC1 proteins were purified from total soluble membrane extract using the HisPur Cobalt Purification Kit (Thermo Scientific), which takes advantage of the high affinity that the polyhistidine tag on the protein has for Co2þ ions. The use of Co2þ chelate resin is preferred for its lower nonspecific binding than that of Ni2þ agarose resin. Figure 5 shows a representative single-channel experiment that resulted from the use of this protocol.

11.2.1 Purification of human TPC1 overexpressed in HEK293 cells 1. HEK293 cells stably overexpressing His-tagged human TPC1 are harvested and resuspended in ice-cold immunoprecipitation buffer (IP) containing 150 mM NaCl, 25 mM Tris, pH 7.4, and supplemented with EDTA-free complete protease inhibitors cocktail (Roche Applied Sciences). 2. Glass beads (Sigma) at a 1:1 (v/v) ratio are added to the cell suspension. Mechanical disruption of the cell membranes is achieved by passing the cells and beads in suspension 20 times through a 23-gauge needle. 3. The glass beads and nuclei are then gently pelleted at 2,000  g for 5 min at 4  C. 4. The resulting postnuclear supernatant is spun at 100,000  g for 1 h at 4  C. 5. The mixed membrane fractions, which are recovered in the pellet, are then subjected to solubilization in the same IP buffer containing 1% CHAPS/0.2% PC for 2e3 h at 4  C under continuous agitation. 6. After incubation, the membrane suspension is centrifuged at 20,000  g for 10 min at 4  C, to remove insoluble material. 7. The Co2þ resin contained in a column is first spun at 700  g for 2 min to remove the storage buffer. The resin is then washed with equilibrium buffer

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8.

9. 10.

11.

(50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.4, supplemented with 0.4% CHAPS/ 0.2% PC) and subsequently spun at 700  g for 2 min. The supernatant is then added to the column at a concentration not exceeding the maximum loading capacity (see HisPur Cobalt Purification Kit instructions for details) and mixed for 2-3 hours at 4 C. The column is washed with equilibrium buffer 3 times to remove unbound proteins. His-tagged human TPC1 proteins bound to the Co2þ resin are then eluted with a buffer containing 50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7.4 plus 0.4% CHAPS/0.2% PC. The eluted fraction is then dialyzed overnight against a solution containing 50 mM sodium phosphate, 300 mM NaCl, pH 7.4. After dialysis, the fraction containing purified TPC1 is mixed in a 1:1 ratio with 0.5 M sucrose and snap frozen in liquid nitrogen.

The resulting purified TPC1 proteins can then be reconstituted into artificial membranes for subsequent investigation of their single-channel properties.

12. DISCUSSION We have presented the most frequently used techniques for examining the singlechannel function of ion channels located on intracellular membranes that we use in our laboratory. However, it is important to recognize that other investigators may choose different variations of the methods described here. Indeed, we are continually refining our own methods of ion channel reconstitution to suit a particular ion channel or line of investigation and we remain forever indebted to those who have taught us all that we know of these techniques.

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Reconstitution of lysosomal ion channels into artificial membranes.

Ion channels that are located on intracellular organelles have always posed challenges for biophysicists seeking to measure their ion conduction, sele...
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