caged neurotransmitters and other caged compounds: design and application

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doi: 10.1101/pdb.top084152 Cold Spring Harb Protoc; George P. Hess, Ryan W. Lewis and Yongli Chen Application Caged Neurotransmitters and Other Caged Compounds: Design and Service Email Alerting click here. Receive free email alerts when new articles cite this article - Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (42 articles) Patch Clamping (255 articles) Neuroscience, general (57 articles) Electrophysiology http://cshprotocols.cshlp.org/subscriptions go to: Cold Spring Harbor Protocols To subscribe to © 2014 Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press at UNIVERSITE LAVAL on October 10, 2014 - Published by http://cshprotocols.cshlp.org/ Downloaded from Cold Spring Harbor Laboratory Press at UNIVERSITE LAVAL on October 10, 2014 - Published by http://cshprotocols.cshlp.org/ Downloaded from

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doi: 10.1101/pdb.top084152Cold Spring Harb Protoc;  George P. Hess, Ryan W. Lewis and Yongli Chen ApplicationCaged Neurotransmitters and Other Caged Compounds: Design and

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CategoriesSubject Cold Spring Harbor Protocols.Browse articles on similar topics from

(42 articles)Patch Clamping (255 articles)Neuroscience, general

(57 articles)Electrophysiology

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© 2014 Cold Spring Harbor Laboratory Press

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Topic Introduction

Caged Neurotransmitters and Other Caged Compounds:Design and Application

George P. Hess, Ryan W. Lewis, and Yongli Chen

The approaches using caged neurotransmitters described here enable transient kinetic investigations tobe made with membrane-bound proteins (receptors) on a cell surface with the same time resolution aswas previously possible only with proteins in solution.

INTRODUCTION

Elegant statistical techniques, including recording single-channel currents (Sakmann and Neher 1983,1995), exist for investigating receptor-mediated reactions on cell surfaces and provide valuable infor-mation about the ion specificity, conductance, and lifetime of the open channel. In single-channelcurrent measurements, the receptors and ligands are in a quasi equilibrium. However, there remaininteresting questions that can be answered if, before initiating receptor-mediated reactions, one canequilibrate ligands with the receptors on cell surfaces in times short compared with channel openingand desensitization, thus greatly improving the temporal resolution of the experiments. This goal canbe accomplished by using photolabile, inert precursors of neurotransmitters (“caged” neurotransmit-ters) that can be equilibrated with cell-surface receptors before photolytically releasing the neuro-transmitter, thus avoiding diffusional barriers. Once equilibrated, the caged neurotransmitter can berapidly cleaved in the microsecond time region by a pulse of light of the appropriate wavelength andenergy, thus releasing free neurotransmitter. Caged compounds can also provide spatial resolution(Li et al. 1997), depending on the area illuminated and the duration of illumination.

Photocleavable protecting groups for biologically important compounds have many uses. This isparticularly true of cases in which access of a compound to its reaction partner is slow, but the inducedreaction is fast (Kaplan et al. 1978; McCray and Trentham 1989; for reviews, see Adams and Tsien1993; Corrie and Trentham 1993; Hess 1993; Nerbonne 1996). Several common caging groups that areused in biological assays include 2-methoxy-5-nitrophenyl (MNP) esters (Ramesh et al. 1993; Niuet al. 1996c), p-hydroxyphenacyl derivatives (Park and Givens 1997), desyl-based compounds (Givenset al. 1998), coumarin esters (Bendig et al. 1997; Furuta and Iwamura 1998), and ruthenium com-plexes (Rial Verde et al. 2008; see also Inorganic Caged Compounds: Uncaging with Visible Light[Zayat et al. 2007]). The basic structure of several of these caging groups can be found in Figure 1.

CAGED NEUROTRANSMITTERS: PHOTOLABILE, BIOLOGICALLYINERT NEUROTRANSMITTER PRECURSORS

Many photolabile protecting groups have been identified and studied for their synthetic properties(Bochet 2002), and several of these groups have become excellent tools for the study of biological

Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011.

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systems (Mayer and Heckel 2006). The most frequently used protecting group is the 2-nitrobenzylgroup, with various substituents (De Mayo 1960; Barltrop et al. 1966; Patchornik et al. 1970; McCrayet al. 1980; Corrie and Trentham 1993). The use of this protecting group was pioneered by Engels andSchlaeger (1977) and for biologically important phosphates by Kaplan et al. (1978) and McCray andTrentham (1989), and led to widespread use of “caging groups”withmany other biological molecules,including neurotransmitters, enzyme substrates, cofactors, nucleic acids, oligonucleotides such asaptamers and siRNAs, specific residues of peptides and proteins, Ca2+, phospholipids, steroids,hormones, and many others (for review, see Mayer and Heckel 2006).

The caging group and the functional group of the neurotransmitter to which it is attached, thephotolysis characteristics, and the by-products of photolysis all play a role in determining whethera caged compound is satisfactory for a particular purpose. This is not, so far, predictable and mustbe determined experimentally. As an example, the αCNB (α-carboxy-2-nitrobenzyl)-caged GABA(γ-aminobutyric acid) and other caged GABA molecules that are satisfactory for use with α1β2γ2LGABAA receptors inhibit α1β2δ GABAA receptors at the same concentration (KP Eagen, GP Hess,unpubl. data).

Experimental Considerations for Caged Neurotransmitters

A systematic approach to the development of a caged compound and to its use in answering inter-esting biological questions is recommended.

1. The quantum yield and rate of photolysis of the caged compound at the wavelength to be usedmust be known. The quantum yield determines the maximum amount of neurotransmitter thatcan be released by photolysis. The photolysis rate determines how fast a reaction can bemeasured.

2. A functional assaymust be used to determine whether the caged compound (before photolysis) orits photoproducts other than the desired compound are biologically inert in the system one wishesto use.

R′

R′

R

NO2

R

LG

NO2

N

LG

O

N

N

N

NLG

O

Coumarinderivatives

Rutheniumderivatives

O

R′

R

Ru

P

LG

HO

2-Nitrobenzylderivatives

7-Nitroindolinederivatives

2-(2-Nitrophenyl)-propylderivatives

ρ-Hydroxyphenacylderivatives

R′′

R′

NO2

R

LG

LG

R′′

FIGURE 1. A noninclusive list of several generic caginggroups that have been used in biological assays. Themost commonly used caging group, or derivativethereof, is the 2-nitrobenzyl group. Both 7-nitroindolineand coumarin derivatives are also widely used. LG,leaving group; R, unspecified functional group.

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3. A method for calibrating the concentration of compound released on photolysis is generallynecessary.

Caged neurotransmitters must meet several important criteria.

1. They must be soluble in aqueous solutions and sufficiently stable at physiological pH beforephotolysis.

2. They must be photolyzed at a wavelength >335 nm to avoid cell damage.

3. Neither the caged compound nor the photolysis products, with the exception of the liberatedneurotransmitter, should modify the receptor-mediated reaction being studied.

For use in transient kinetic experiments, caged neurotransmitters must also meet the followingcriteria.

4. Theymust be photolyzed in themicrosecond time region so that photolysis is not rate limiting.

5. They must photolytically release the neurotransmitter with sufficient quantum yield to allowkinetic investigations to be made over a wide range of neurotransmitter concentration.

The quantum yield of a caged compound can be measured with several techniques, the simplestof which is photolysis of a sample of a caged molecule with pulses of light of known energy whilemonitoring spectroscopic changes in the absorption or fluorescence spectrum (Milburn et al. 1989). Ifspectroscopic changes are not observed, an alternative method is to analytically separate and quantifythe caged and uncaged molecules by techniques such as high performance liquid chromatography(HPLC) (Milburn et al. 1989).

The rate of photolysis can be approximated from the data obtained in a quantum yield determi-nation if the duration of a single light pulse is known. It can also be determined by measuring the rateof change of any observed transient absorption that occurs during photolysis, a method that isdescribed in the literature (Walker et al. 1986, 2002; Milburn et al. 1989). All these criteria werekept in mind when we initiated the synthesis of photolabile inert precursors of neurotransmitters. Wetried using derivatives of the 2-nitrobenzyl group to cage carbamoylcholine (Walker et al. 1986), astable and well-characterized analog of acetylcholine with an amino group. The initial compoundswere not suitable for rapid kinetic investigations because they were not biologically inert (Walker et al.1986) or they photolyzed too slowly. However, when we introduced the use of α-CNB (Milburn et al.1989) to protect the carboxyl group of neurotransmitters, we obtained compounds that meet all thecriteria listed above for transient kinetic investigations of the acetylcholine, glutamate (Wieboldt et al.1994), kainate (Niu et al. 1996a), and GABA (Gee et al. 1994) receptors. In the case of the neuro-transmitter glycine, we used the 2-methoxy-5-nitrophenol protecting group (MNP), creating a de-rivative that is photolyzed in the 1-msec time region but that is not stable in aqueous solution(Patchornik et al. 1970; Ramesh et al. 1993). So, we turned to β-alanine, which also activates theglycine receptor (Choquet and Korn 1988), to make an MNP-caged β-alanine that has all the desiredproperties for transient kinetic investigations of the glycine receptor (Niu et al. 1996c). It is importantto note that if the ligand to be “caged” absorbs light at the same wavelength as the caging group,problems may be encountered (Breitinger et al. 2000).

Visible light-sensitive photolabile neurotransmitters have several advantages compared to theirUV-sensitive counterparts because visible light is less damaging to the cells/receptors and becausevisible light flash lamps are more affordable than lasers. An additional advantage is that cagedcompounds photolyzable with visible light can be used for two-photon microscopy (Denk 1994).The neurotransmitters glutamic acid and glycine were initially caged (Shembekar et al. 2005, 2007)with a coumarin derivative that had been used previously to cage cAMP and cGMP (Hagen et al.2001). The coumarin-caged glutamic acid and glycine can be photolyzed in the microsecond timeregion by visible light with a good quantum yield and are suitable for transient kinetic investigations(Shembekar et al. 2005, 2007). Several other visible light-sensitive compounds have been reportedusing various caging groups, including ruthenium complexes (Rial Verde et al. 2008), coumarinderivatives (Fan et al. 2009), and a 2-nitrobenzyl derivative (Banerjee et al. 2003).

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Purification and Storage of Caged Compounds

Contamination of a caged compound by a small amount of the uncaged compound is one of the mostfrequently encountered problems in their use. Small amounts of uncaged neurotransmitter candesensitize the receptors during equilibration with the caged compound before photolysis. Toavoid these problems, every caged neurotransmitter must be tested for purity (even if obtainedfrom a commercial source), purified if necessary, and stored appropriately. The inertness of cagedneurotransmitters we use are tested with the cell-flow technique (Udgaonkar and Hess 1987) de-scribed below. Testing the caged neurotransmitter in this manner easily identifies if a solution of cagedcompound is contaminated by free neurotransmitter that will activate the receptor. We find that thismethod is much more sensitive for detecting low concentrations of neurotransmitter than can bemeasured by separation techniques such as TLC, HPLC, and the like. If necessary, the caged com-pounds are purified using various chromatography techniques. Purification details are given in thepertinent references for each compound.

To avoid degradation of caged compounds and the generation of free neurotransmitter, precau-tionsmust be taken to protect the compounds from light, and they should be stored over a desiccant at−20˚C to −80˚C. Occasionally it is found necessary to work under controlled lighting conditions,choosing the lighting relative to the caged compound (e.g., red lamps for compounds sensitive tovisible light). We store caged compounds protected from light in brown vials or amber Eppendorftubes, wrapped in black electrical tape or aluminum foil. To avoid releasing free neurotransmitter as aresult of hydrolysis, solutions of caged compounds should be prepared immediately before use.Thermal stability is also an issue for some compounds, in which case the solutions should be kepton ice until used.

CELL-FLOW TECHNIQUE

The cell-flow technique (Hess et al. 1987; Udgaonkar and Hess 1987) can be used for testing biologicalinertness of a caged neurotransmitter, calibrating concentration of free neurotransmitter released byphotolysis, and checking cell viability. It involves (i) the whole-cell current-recording technique(Hamill et al. 1981; Marty and Neher 1995), which allows one to determine the current arising fromopen receptor channels on the cell surface at constant voltage; (ii) a U-tube device (Krishtal andPidoplichko 1980; Udgaonkar and Hess 1987) that allows solutions containing neurotransmitter toflow over a cell; and (iii) when necessary, a method to correct the current amplitude for receptordesensitization that occurs while the neurotransmitter equilibrates with the receptors (Hess et al. 1987;Udgaonkar andHess 1987). In the accompanying protocol,Cell-FlowTechnique (Hess et al. 2014), wedescribe the construction of the U-tube device and the whole-cell current-recording technique.

The cell-flow method is used for several purposes.

1. The first is to determine that the caged neurotransmitter itself is not an agonist or inhibitor of thereceptor and that the receptor is not modulated by the protecting group after photolysis. This isdone by determining the whole-cell current in (i) cell-flow measurements using a standardconcentration of neurotransmitter, (ii) the same measurement in the presence of a large excessof caged neurotransmitter, and (iii) the same measurements after the cell has been exposed tocaged neurotransmitter for several seconds.

2. During photolysis experiments the stability of the whole-cell patch may begin to deteriorate,causing a change in the current response of a cell to a standard agonist concentration. As a controlmeasurement to determine the integrity of the whole-cell patch, a standard neurotransmitterconcentration, typically a saturating concentration, is applied to cells before and after photolysismeasurements. Equivalent responses to the standard solution verify that the receptors and whole-cell patch have not changed over the course of an experiment. If a change is detected, all themeasurements after the initial control measurement are discarded and a new cell is used. Whole-cell patches in cell-flow experiments are typically stable enough that three independent cells may

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be sufficient to construct a full dose-dependent response curve covering a wide range of neuro-transmitter concentration (Fig. 2D).

3. Neurotransmitter dose-dependent curves obtained with the cell-flow technique are needed forcalibrating the concentration of neurotransmitter generated by photolysis of a caged precursor inmeasurements using the laser-pulse photolysis technique (Milburn et al. 1989; Matsubara et al.1992; Hess and Grewer 1998). These estimates are obtained by comparing current responses froma known concentration of free neurotransmitter and the amplitude of the current evoked on thephotolysis of the caged compound, together with a dose–response curve.

Whole-Cell Current-Recording Technique

Whole-cell patch-clamping procedures are thoroughly explained in Hamill et al. (1981), Marty andNeher (1995), Sakmann and Neher (1995), and Walz et al. (2002). We describe how we measurewhole-cell currents in the accompanying protocol, Cell-Flow Technique (Hess et al. 2014).

The U-Tube Cell-Flow Device

Various flow devices have been used to flow neurotransmitter solutions over cells containing receptors(Krishtal and Pidoplichko 1980; Trussell and Fischbach 1989; Vyklicky et al. 1990; Franke et al. 1993;Edmonds et al. 1995). Problems are encountered with some devices, such as piezo-electric translatorsand theta tubes, and multibarreled tubes. With many of the devices, the orientation between theporthole of the flow device and the cell is notmaintained absolutely constant. Orientation is critical forreproducibility in kinetic experiments (Hess et al. 1987; Udgaonkar and Hess 1987; Niu et al. 1996b).

To be able to change the composition of the flowing solution during an experiment and stillmaintain a constant orientation, wemodified (Niu et al. 1996b) the design of Krishtal and Pidoplichko(1980) and use stainless steel tubing. A diagram of our U-tube cell-flow device is shown in Figure 2Band its construction is described in the accompanying protocol, Cell-Flow Technique (Hess et al.2014). The porthole has a diameter of �150 µm and is placed �100–200 µm from a cell suspendedfrom the recording electrode. In brief, the neurotransmitter solution emerges from the porthole of thedevice at a linear velocity of 1–3 cm/sec. A more rapid flow is a disadvantage because the integrity ofthe whole-cell seal between the cell and the electrode tends to deteriorate, and fewer measurementscan be made with each cell.

One must also use a laminar flow of solution and avoid turbulence. Accordingly, the cell must besuspended in the flowing solution andmust be nearly spherical. This is accomplished after making thewhole-cell seal between the recording electrode and a cell by lifting the cell from the bottom of theculture dish so that the cell is suspended from the recording electrode (Udgaonkar and Hess 1987).

Alternatively, vesicles or patches of�10-µm diameter can be pulled from the cell. Patch formationis described in detail in Sakmann and Neher (1995). The method for obtaining vesicles has beendescribed by Walstrom and Hess (1994) and is similar to that described by Sather et al. (1992). Inbrief, a vesicle is obtained from a cell body by first making a whole-cell seal (Hamill et al. 1981) andthen gently lifting the recording pipette until the membrane pinches off from the cell body, thusforming a vesicle. The vesicles typically have a diameter of �10 µm and a capacitance of �1–3 pF. Adisadvantage of membrane patches is that the receptor concentration is considerably lower than in awhole cell, and consequently measurements must be made with more patches to achieve satisfactorystatistics.

Correcting the Observed Whole-Cell Current for Receptor Desensitization

If the receptor being studied desensitizes during the current increase time, the amplitude of themaximum current recorded can be corrected to take into account the desensitization (Udgaonkar1986; Hess et al. 1987). The correction is based on theories of solution flow over submerged sphericalobjects (Landau and Lifshitz 1959; Levich 1962) and on the observation that many cells becomespherical when detached from the substratum. At the flow rates we use, 1–4 cm/sec, the rate-limiting

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10

11

12

1

9

8

3

7

4

A B

5

6 Directionof flowingsolution

U-tube

200 μmOpticalfiber

Cell100 μm

25 μm150 μm

Referenceelectrode

Recordingpipette

2

C D2.5

2.0

1.5

1.0

0.5

0.0–250 1650 3550 5450 7350 9250

Time (msec)

Who

le-c

ell c

urre

nt (

nA)

5

4

3

2

1

010–2 10–1 100

Carbamoylcholine (mM)

I A (

nA)

101

E Laser

–800

Cur

rent

(pA

) –600

–600

Flash lamp

–400

–400

Cur

rent

(pA

)

–200–200

0 0

–2 0 2 4 6 8 20 40 60 80

Time (msec)

–2 0 2 4 6 8 20 40 60 80

Time (msec)

FIGURE 2. (A) A schematic drawing of the device used for the flash/laser-pulse photolysis technique. The componentsshown include the stainless steel U-tube (1), the fiber optic cable (2), a borosilicate recording pipette containingintracellular buffer and the recording electrode (3), the pipette holder (4), the head stage (5), the suction/vacuum tube(6), the reference electrode (7), the microscope (objective) for viewing cells and aligning the U-tube (8), a cell cultureplate with cells expressing the receptor of interest (9), a three-port solenoid valve (10), 0.38- or 0.42-mm inner-diameter peristaltic tubing drawing solution away from the U-tube (11), and 0.25- or 0.5-mm inner-diameter peristaltictubing with solution flowing toward the U-tube (12). The cell-flow technique requires all of the same components,omitting only the optical fiber. (B) A magnified diagram depicting the alignment of components needed for the flash/laser-pulse photolysis technique. While the solenoid valve is open, solution is actively drawn away from the U-tube ata higher rate than that at which solution flows into the U-tube. The U-tube draws extracellular buffer in through theporthole from the dish, preventing any leakage or diffusion of ligand solution over the cell. When the solenoid valvecloses, solution being pumped to the U-tube is forced out of the U-tube porthole and over the surface of the cell. Linearflow rates of 1–4 cm/sec are typically used. (C ) Cell-flowmeasurement with a BC3H1muscle cell containing nicotinicacetylcholine receptors, pH 7.4, 23˚C, −60 mV transmembrane potential. A 200-µM acetylcholine solution emergedfrom the cell-flow device (A) at a rate of 1 cm/sec. (Thick solid line) The observed current; (thinner line) the calculatedcurrent corrected for receptor desensitization. (Reprinted from Hess et al. 1987.) (D) Concentration dependence of thecurrent amplitude corrected for receptor desensitization, IA. BC3H1 muscle cells, pH 7.4, 22˚C–23˚C, and −60 mVtransmembrane potential. Data are from Udgaonkar and Hess (1987) (•, single–channel current recordings; ▴, cell-flow measurements) and Matsubara et al. (1992) (□, laser-pulse photolysis). The line indicating the concentration ofopen receptor channels was calculated from the constants pertaining to the channel-opening process determined inlaser-pulse photolysis experiments (Matsubara et al. 1992). Reprinted from Hess and Grewer (1998). (E) Whole-cellcurrents induced by the photolytic release of �100-µM glutamate from 2 mM coumarin-caged glutamate using a laser(left) or a flash lamp (right) were recorded from HEK 293 cells stably transfected with cDNA encoding GluR6 kainatereceptors (Y Chen, GP Hess, unpubl. data). The bath buffer contained 150 mM NaCl, 10 mM HEPES, and 1 mM CaCl2.The pipette buffer contained 120 mM CsCl, 10 mM HEPES, and 10 mM EGTA. The buffers were adjusted to pH 7.4using NaOH and CsOH, respectively. The experiments were performed at room temperature and a clamped voltageof −60 mV.

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step in equilibration of ligand with the cell surface is the velocity of a layer of the solution (whichbecomes the diffusion boundary�2 µm above the cell surface [Levich 1962]) emerging from the flowdevice and from which the ligand diffuses to the cell surface. The buildup in ligand concentration onthe cell surface below this solution layer is rapid (2–5 msec), depending on the flow rates used(Udgaonkar 1986; Hess et al. 1987). Knowing the ligand concentration on the cell surface allowsone to correct the observed current during the current increase for receptor desensitization that occursduring themeasurement (Udgaonkar 1986; Hess et al. 1987). Receptor desensitization is characterizedby the rate coefficient a and can be measured independently in each experiment (Fenwick et al. 1982;Clapham andNeher 1984). The corrected current, IA, is defined as the amplitude of the current arisingfrom receptors on the cell surface in the absence of desensitization and at a definite ligand concen-tration (Udgaonkar 1986; Hess et al. 1987). To obtain the value of IA from measurements of theobserved current Iobs, we divide the current time course into constant (�5-msec) time intervals to takeinto account the equilibration time of small segments of the cell surface with ligand as the solutionflows from the U-tube over the cell. The current is then corrected for the desensitization occurringduring each time interval Δt. After n constant time intervals (nΔt = tn), during each of which thecurrent (Iobs)Δt is measured, the corrected current is given by (Udgaonkar 1986; Hess et al. 1987)

IA = (eaDt − 1)∑

(Iobs)Dti + (Iobs)Dtin, (A)

where (Iobs)Δti is the observed current during the ith time interval and tn is equal to or greater than thecurrent increase time (Udgaonkar 1986; Hess et al. 1987). The value of IA was found to be independentof the solution velocities used in the cell-flow method and can be determined with good precision(±10%) (Udgaonkar 1986; Hess et al. 1987). The observed current in a cell-flow experiment (solidline) and the current corrected for desensitization (thin line) are shown in Figure 2C.

The relationship between IA and the concentration of receptors in the open-channel form in theabsence of desensitization is given by Equation 1B in Table 1. IM represents the current produced by1 mol of open receptor channels, RM the moles of receptors on the cell surface, and (AL2)o the fractionof receptors present that are in the open-channel form. In terms of the scheme at the head of Table 1(Eq. 1), (AL2)o is given (Cash and Hess 1980) by Equation 1A in Table 1.

Using the nicotinic acetylcholine receptor in BC3H1 cells as an example, Figure 2D shows thedependence of IA [�(AL2)o] on carbamoylcholine concentration over a 500-fold range. The solidtriangles represent results obtained by the cell-flow technique after the current was corrected fordesensitization. The circles show the dependence of (AL2)o on the carbamoylcholine concentrationwhen determined by an entirely different approach and methodology. The probability P0 that thechannel is open while the receptor is in a nondesensitized state (Neher 1983; Ogden and Colquhoun1985) was determined at three carbamoylcholine concentrations. The P0 values were obtained fromsingle-channel current measurements (Neher and Sakmann 1976) and represent the fraction of timethe channel is open while the receptor is in a nondesensitized state (Eq. 1A in Table 1). The opensquares represent results obtained by the laser-pulse photolysis technique (see below) (Milburn et al.1989; Matsubara et al. 1992; Hess and Grewer 1998), the time resolution of which is sufficient so thatthe observed current obtained using the whole-cell current-recording technique does not have to becorrected for desensitization. The agreement between the results obtained using the three differenttechniques confirms the validity of the approach.

See the protocol Cell-Flow Technique (Hess et al. 2014) for additional details on how we measurewhole-cell currents.

PHOTOLYSIS TECHNIQUE

Kinetic measurements and localization studies can be performed by using the photolysis technique.The apparatus used to perform these measurements is depicted in Figure 2A and includes thewhole-cell current-recording instrumentation that we use for the cell-flow technique, as well as a

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TABLE 1. Equations used in analysis of the mechanism of the nicotinic acetylcholine receptor in BC3H1 cells

A+ L QRK1 AL2 QR

kop

kclAL2 (1)

(AL2)o =AL2

A+ AL+ AL2= L2

(L+ K1)2F+ L2= Po. (1A)

In Equation (1A), A and AL2 represent the receptor in the closed-channel form, and (AL2)o is the fraction of nondesensitizedreceptor molecules in the open-channel form. Φ−1 = kop/kcl is the channel opening equilibrium constant; kop and kcl are therate constants for channel opening and closing, respectively. L represents the molar concentration of activating ligand, and K1

is the dissociation constant of the neurotransmitter from the sites controlling channel opening. Po is the conditionalprobability, determined in single-channel current recordings, that the receptor is in the open-channel form (Udgaonkarand Hess 1987).

IA = IMRM(AL2)o. (1B)In Equation (1B), IA is the current due to open receptor channels in the cell membrane corrected for receptor desensitization,IM is the current due to 1 mol of open receptor channels, and RM represents the number of moles of receptor in the cellmembrane. Equation (1C) is a linear version of Equation 1B (Hess et al. 1983):

[IMRM(IA)−1−1]1/2 = F1/2 +F1/2K1[L1]−1. (1C)The desensitization phase of the observed current is described by Equation (2A):

(Iobs)t − (Iobs)t=1 = [(Iobs)t=0 − (Iobs)t=1]e−at. (2A)Here α represents the rate coefficient for receptor desensitization obtained from the falling phase of the current in cell-flowexperiments (Udgaonkar and Hess 1987). Iobs represents the current during the falling phase. The subscripts t and (t = 0) referto the time of measurement; (t =∞) refers to the time when an equilibrium between active and desensitized forms has beenreached. The exponential parameter α is described as

a = FL k43 + 2K2k21

(L+ 2K2) + (L2k34 + 2K1k12)LL2(1+F) + 2K1LF+ K2

1F

[ ]. (2B)

Here k12, k34 and k21, k43 represent the rate constants for desensitization and resensitization, respectively. When k34 is thedominant rate constant, a simplified equation is obtained (Udgaonkar and Hess 1987):

a = k43FL2

(L+ K1)2F+ L2. (2C)

The dissociation constant of the inhibitor from the nondesensitized receptor can be determined by both cell-flow andphotolysis measurements. To simplify the equations, we use a ratio method, IA/IA′ where IA and IA′ represent the currentmaxima corrected for receptor desensitization in the absence and presence of inhibitor, respectively. The relationshipbetween the observed inhibitor dissociation constant K and the inhibitor dissociation constant for the A, AL, AL2, and AL2receptor forms FA, FAL, FAL2 , and FAL2

, represent the fraction of receptors in forms A, AL, AL2, and AL2. I0 and II0 represent the

concentrations of two different inhibitors, and KI and KII are the observed dissociation constants.

IA/IA′ = 1+ I0/K1, (3A)

1K1

= FA(K1)1

+ FAL(K1)2

+ FAL2(K1)3

+ FAL2(K1)4

, (3A′)

IAIA

′ = 1+ I0K1

+ (2K1L+ K21 )F

(L+ K1)2F+ L2. (3B)

Equation (3B): For a competitive inhibitor, 1/KI is multiplied by the fraction of receptor molecules in the A form and theAL form.

IAIA

′ = 1+ I0K1

(AL2)o. (3C)

Equation (3C): An inhibitor binding only to the open-channel form.

IAIA

′ = 1+ I0KI

II0KII

. (3D)

Equation (3D): Two inhibitors, I0 and II0, binding to the same receptor site.

(continued )

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fiber-optic cable and a light source. Photolysis can be performed using either a laser or a suitablelight source.

Laser-Pulse Photolysis Technique

A variety of different lasers can be used as light sources for photolysis, depending on whichmodels areavailable. The advantage of a dye laser is the relatively long laser pulse length (500 msec); experimen-tally we find that the cells are less stable when pulse lengths shorter than 600 nsec are used. Whateverthe light source, the light beam is introduced from an optical fiber of 200- to 400-µm (core) diameter(Newport) depending on the amount of energy needed to be delivered.We typically adjust the amountof energy of a single pulse of laser light emerging from the fiber to�500 µJ, as determined with a Joulemeter (model ED-200, Gentec, Palo Alto, CA).

Light from a tungsten source (Newport 780) can also be projected through the optical fiber.Although not essential, this allows one to position the fiber, which also carries the laser light, sothat the cell is in the center of the beam of laser light. The irradiated area is adjusted by positioning theoptical fiber above the cell surface to have a light diameter between 300 and 400 µm. This area issufficient so that the concentration of photoliberated neurotransmitter is constant during the timeinterval of the kinetic (current) measurements.

The cell attached to the current-recording electrode is equilibrated with caged compound. At time0 a single pulse of laser light photolyzes the caged compound in the microsecond time region. Theliberated neurotransmitter binds to the receptors on the cell surface and initiates the formation oftransmembrane channels. The time resolution of the technique allows one to observe three distinctphases of the reaction: a rising phase of the current reflecting the opening of acetylcholine receptorchannels, maximum current amplitude, a measure of the concentration of open receptor channels,

TABLE 1. Continued

IAIA

′ = 1+ I0KI

+ II0KII

+ I0KI

II0KII

= 1+ I0KI

+ II0KII

KI + I0KI

( ). (3E)

Equation (3E): Two inhibitors, I0 and II0, binding to two different receptor sites.

It = Imax[1− exp(−kobst)]. (4A)Equation (4A): In the laser-pulse photolysis experiments with BC3H1 cells containing nicotinic acetylcholine receptors, thecurrent increase timewas observed to follow a single, exponential rate law over 85%of the reaction (Matsubara et al. 1992). Inthis equation, It represents the observed current at time t and Imax the maximum current. kobs is the first-order rate constant forthe current increase.

kobs = kcl + kopL

L+ K1

( )2

. (4B)

Equation (4B): The relationship between the observed rate constant for the current increase kobs, and kop, kcl, K1 of the reactionscheme (see the beginning of this table).

kobs = kcl + K1

K1 + I0+ kop

LL+ K1

( )2

. (4C)

Equation (4C): kobs in the presence of an inhibitor that binds only to the open-channel form of the receptor.

kobs = kcl + kL

L+ K1

( )2 K1

K1 + Io. (4D)

Equation (4D): kobs in the presence of an inhibitor that binds only to the closed-channel form of the receptor. If the inhibitorbinds both to the open- and closed-channel forms, a combination of Equations 4C and 4D is obtained.

Reprinted from Hess and Grewer 1998.

The equations are based on the assumption that the binding of two ligand molecules (acetylcholine or carbamoylcholine) is required beforethe opening of the transmembrane channel (Katz and Thesleff 1957; Reynolds and Karlin 1978; Hess et al. 1983). A, AL, and AL2 representreceptor forms with none, one, or two ligand molecules bound, and AL2 represents the open-channel form of the receptor (with two ligandmolecules bound).

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and on a different and slower timescale, the falling phase of the current reflecting receptor desensi-tization. In experiments with the excitatory acetylcholine, glutamate, kainate receptors, and theinhibitory GABA and glycine receptors (Milburn et al. 1989; Matsubara et al. 1992; Ramesh et al.1993; Gee et al. 1994;Wieboldt et al. 1994; Niu et al. 1996c), the increase time of the current followed asingle exponential rate equation over 85% of the reaction.

The dependence of the first-order rate constant for the current increase time is plotted versuscarbamoylcholine concentration according to Equation 4B in Table 1. The ordinate intercept of theline gives the value of the channel-closing rate constant kcl, and the slope allows evaluation ofthe channel-opening rate constant kop. From the dependence of the maximum current amplitudeon the concentration of carbamoylcholine, one can obtain the value of the channel-opening equilib-rium constant Φ (= kop/kcl), and the value of K1, the dissociation constant of the receptor sitecontrolling channel opening, by using Equation 1C in Table 1. The values of K1 and Φ obtainedfrom the effect of ligand concentration on the current amplitude can be compared to the values ofK1, kcl, and kop obtained from the effect of ligand concentration on the observed rate constant kobsfor the increase time of the current (Table 1, Eq. 4B). The falling phase of the current gives infor-mation about the rate of desensitization. When present, receptor desensitization is slow comparedto channel opening, occurs in a different timescale, and is investigated more conveniently by thecell-flow method (Udgaonkar and Hess 1987).

The laser-pulse photolysis technique allows one to determine the rate constant of the currentincrease indicative of channel opening and, therefore, to study the effects of inhibitors on kcl (Table 1,Eq. 4C) and kop (Table 1, Eq. 4D) independently of one another.

The time resolution of the kinetic methods just described allows one to determine the maximumcurrent amplitudes and, therefore, the concentration of open receptor channels before desensitizationoccurs. This, in turn, allows one to determine the effects of inhibitors or potentiators (or mutationsthat modulate receptor current) on the current amplitudes at low concentrations of neurotransmitterwhen the receptor is mainly in the closed-channel form, and at high concentrations when the receptoris mainly in the open-channel form (Niu and Hess 1993; Niu et al. 1995; Ferster et al. 1996; Hess andGrewer 1998). These measurements allow one to differentiate between noncompetitive and compet-itive inhibitors (Table 1, Eqs. 3A and 3B) and to determine whether two noncompetitive inhibitorsbind to two different sites (Table 1, Eq. 3E) or to the same site (Table 1, Eq. 3D).

Flash-Lamp Photolysis with Visible Light

Originally, suitable caged neurotransmitters were photolyzed in the near-UV region using lightfrom a laser (for review, see Marriott 1998; Goeldner and Givens 2005). In an effort to make transientpre–steady state kinetic studies of reactions mediated by ligand-gated ion channels more widelyaccessible and for use in multiphoton microscopy (Matsuzaki et al. 2001; Smith et al. 2003; Trigoet al. 2009), we embarked on the development of caged neurotransmitters that can be photolyzed byvisible light.

A light source appropriate for such studies must meet certain requirements. Light of a suitablewavelength, delivered from the exit of an optical fiber to a cell in the whole-cell current-recordingmode, must be of sufficient energy in a single pulse to release free neurotransmitter but not of suchenergy as to damage the protein or cell. For transient kinetic studies, the pulse durationmust be withinthe microsecond domain. A variety of visible light sources were considered. Engert et al. (1996) built alow-cost nitrogen laser with a 5-nsec pulse for flash photolysis of caged compounds. However, al-though the entire pulse energy was�250 µJ, only 20 µJ reached the sample, which is too low to photo-release sufficient concentrations of neurotransmitter to activate receptors in transient kinetic studies.A light-emitting diode (LED) has been used to study intracellular calcium homeostasis (Bernardinelliet al. 2005). However, with a 100- to 1000-msec pulse, it is not suitable for studying receptor activationin the microsecond–millisecond time domain. Xenon flash lamps have been used with caged com-pounds to map functional neuronal connections (Matsuzaki et al. 2001), but in those cases, the goalwas high spatial resolution; the temporal resolution was not sufficient for transient kinetic studies.

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The use of flash lamps for photolysis of caged compounds has been reviewed in detail (Rapp 1998).An XF-10 xenon flash lamp (Hi-Tech Scientific, UK) has been used to photo-release Ca2+ from DM-nitrophen and from nitr-5–caged Ca2+ (Hardie 1995), but it has a pulse duration of 200 msec, which istoo long for transient kinetic measurements. Canepari et al. (2001) used light at 290–370 nm with apulse length of 1 msec from a Rapp xenon flash lamp, a duration suitable for kinetic measurements,to photo-release glutamate, glycine, and GABA from a 7-nitroindolinyl (NI)–caged precursor or4-methoxy-7-nitroindolinyl (MNI)–caged glutamate. The wavelength range of the Rapp xenon flashlamp extends into the visible wavelength region, making it suitable for transient kinetic investigations.

An SP 450 385- to 450-nm band-pass filter is used with the flash lamp. The coumarin-cagedcompounds do not absorb light >500 nm. Other wavelengths can be used by changing the filtercombination. The wide (200- to 1100-nm) spectrum of the lamp allows many filter combinationsto be used to achieve a desired wavelength range.

The pulse length can be adjusted from 2 to 400 µsec by changing the lamp capacitors. A single 220-µsec pulse provides sufficient energy (�350 µJ) to photo-release free neurotransmitter. A Joule meter(Molectron) is used to measure the energy of the light pulse emerging from the quartz fiber. Theavailable short pulse length (2–400 µsec) and tunable high pulse energy (up to 150 J/pulse) make theSP-20 flash lamp system a good light source for use in transient kinetic measurements of reactionsmediated by neurotransmitter receptors. A separate shutter system is not needed because the dataacquisition software used, pClamp, controls the flash-lamp system operation directly via the built-inexternal trigger of the SP-20 flash lamp. In any case, the shutter systems available are too slow for usein transient kinetic measurements.

To minimize light exposure before photolysis is initiated, the Faraday cage surrounding thecurrent recording instrumentation is covered with aluminum foil, and the tubing that is used todeliver the caged-glutamate solution to the cells via a peristaltic pump is also wrapped with foil.Overhead lights should be turned off. If necessary, a red light can be used to illuminate the room.Measurements are made as described in the preceding section, Laser-Pulse Photolysis Technique.

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