72 H. Ishibashi et al.loading specific compounds into cells, or careful quantification ofion channel selectivity profiles. However, this intracellular dialysisis a double-edged sword: Soluble components that influencecellular excitability and contribute to signaling pathways are dialyzedfrom the cell. One frequently encountered consequence is thatreceptor-mediated responses and/or ionic currents that requiresoluble second messengers are absent or rapidly “run down” duringconventional whole-cell recordings (3, 4). Furthermore, intracel-lular ion concentrations are disrupted, hampering efforts to deter-mine physiologically relevant responses or properties of channelsand receptors (5, 6).The problems of cell dialysis have been effectively overcomewith the perforated patch-clamp whole-cell (PPWC) technique.With this approach, a membrane-perforating agent is used toprovide access to the cell interior without mechanical rupture ofthe membrane attached to the patch pipette. The idea was initiatedby Lindau and Fernandez (3), who used ATP (~0.5 mM) topermeabilize the patch membrane and record the passive electricalproperties of mast cells during antigen-induced degranulation, aresponse that was absent in cells dialyzed during conventionalwhole-cell recordings. The access resistances (0.1–5.0 GW) werevery high, however, and the use of ATP as a perforant is limited tomembranes containing ionotropic P2X receptors. An improvementwas the use of the antibiotic nystatin, which provided lower(18–50 MW) and stable access resistance (4). Much wider applica-bility of the technique has resulted from use of this and otherperforants (described below) that can spontaneously form channelsin numerous membranes and result in access resistances that underthe most optimal conditions may approach values comparable tothose obtained with conventional whole-cell recordings.Table 4.1 compares a number of commonly used perforants (seealso Fig. 4.1a). Nystatin (4) and amphotericin B (11) are structur-ally similar antibiotics derived from streptomycetes (Streptomyces)bacteria. They contain a large, amphiphatic polyhydrophylic lactonering that allows them to form aqueous pores in the cell membrane.The properties of these polyene antibiotics have been well charac-terized in lipid bilayers. Their effect on membrane conductance(reflecting their stability as membrane pores) is highly dependenton the antibiotic concentration, membrane composition, andtemperature (7, 22). For example, sterols in the membrane enhancemembrane conductance, and increasing temperature reduces it.The ion permeation properties of the two agents are similar(Table 4.1), both being modestly selective for monovalent cations4.2. Comparisonof PerforantsCommonlyEmployed
734 Perforated Patch ClampTable 4.1Characteristics of perforants used in perforated patch-clamp recordingsPerforant Nystatin Amphotericin B Gramicidin b-EscinClass Polyene antibiotic,StreptomycesnouseiPolyene antibiotic,StreptomycesnodosusLinear polypeptide antibi-otic, Bacillus brevisaSaponinderivativePore structure Lactone ring,interacts withsterols to forms abarrel-like poreLactone ring,interacts withsterols to forms abarrel-like pore15 Alternating dl-aminoacids in b-sheet asend-on-end dimer?Pore radius ~0.4–0.5 nmb~0.4–0.5 nmb£0.35 nmc>10 kDaSelectivity Weakly cationicd(PK:PCl = 30:1)(PK:PCl = 40:1)Weakly cationicd(PK:PCl = 30:1)K+ ³ Na+ ³ Li+Highly cationic,dPCl~0Cs+ ³ K+ ³ Na+ ³ Li+ > Tris+Nonselective,dCa2+permeableConductance ~5 pSe~6 pSe~50 pSfH+ > Cs+ > K+ > Na+ > Li+?Stock conc.gMethanol(5–10 mg/ml)DMSO (25–50 mg/ml)DMSO (25–100 mg/ml)Methanol (10 mg/ml)DMSO (2–50 mg/ml)H2O(25–50 mM)Final conc.g20–400 mg/ml 25–250 mg/ml 5–100 mg/ml 25–50 mMKey refs. (4, 7–10) (7, 10–12) (5, 12, 13) (14, 15)aGramicidin used in most laboratories is commonly gramicidin D, a combination of gramicidin A, B, and C.bInferred from lipid bilayer experiments with nystatin or amphotericin applied to both sides of the lipidbilayers (and different from the channel from one-sided application as used for PPWC recordings).cMyers and Haydon (16) reported that tetramethylammonium (diameter 0.7 nm) was impermeant.dThe selectivity of the polyene antibiotics has not been quantified, although a value of 10:1 appears in theliterature. The values cited here are calculated using the Goldman–Hodgkin–Katz equation and the~50 mV potential across nystatin-doped bilayers in a tenfold KCl activity gradient (17, 18), and from the15- to 16-mV shift in Vrevshown in Fig. 4.3 of (4) in a twofold KCl gradient, with concentrations con-verted to activities. b-Escin is likely nonselective. Permeability sequences are from (17) for amphotericin Band from (16, 19) for gramicidin.eConductance values from lipid bilayers in symmetrical 2 M KCl (nystatin (17); amphotericin (20)). Lower(<1 pS) values would be predicted in physiological ionic strength (20).fConductance values are from lipid bilayers in symmetrical 2 M KCl (21). In approximate physiologicalionic strengths (0.1 M), conductances were about 10 pS (KCl) and 5 pS (NaCl).gA range of values are given in the literature (including those cited under key references) and are based onpersonal experience. For nystatin and gramicidin, DMSO and methanol can be interchanged. Little infor-mation is published about concentration dependence. Tajima et al. (19) noted less frequent perforation(and higher Ra) as gramicidin was lowered from 100 mg/ml, although Kyrizos and Reichling (13)reported more frequent spontaneous membrane rupture at >20 mg/ml.
74 H. Ishibashi et al.and impermeant to divalent ions (17); they also have low singlechannel conductance and a pore diameter small enough to excludemolecules larger than glucose (~0.8 nm) or sucrose (~1 nm).Remarkably, the permeation properties of the amphotericin B andnystatin pores change to predominantly anion-selective whenadded to both sides of the membrane (18). Fortunately, however,their lateral diffusion is limited by the seal (23), and access fromthe interior membrane side does not occur in PPWC recordings(unless the membrane ruptures).The modest ion selectivity of these antibiotics results ineventual equilibration of all intracellular monovalent ions with thepipette concentrations. In contrast, the use of the highly cation-selective linear polypeptide antibiotic gramicidin as the perforantresults in preservation of the physiological intracellular Cl−concen-tration (5, 12). Gramicidin has negligible permeability to multivalentFig. 4.1. Overview of cell perforation. (a) Relative size and selectivity of commonly usedperforants. (b) Current responses to a −5 mV voltage pulse (DV) during the stages of sealinga patch pipette to a cell to form a GW seal (upper drawing ) followed by subsequentperforation of the patch membrane and cancelation of the current transients (lower traces ).The recordings were obtained using an acutely isolated hippocampal neuron.
754 Perforated Patch Clampions and monovalent anions under physiological conditions in bothbilayers and cells (Table 4.1) (16, 19). Although it has a minimalpore diameter similar to those of amphotericin B and nystatin, ithas much larger single-channel conductance. Gramicidin PPWCrecordings have resulted in a greater understanding of Cl−homeo-stasis in cells under various conditions and of the physiologicalresponses of Cl−permeant ionotropic g-aminobutyric acid (GABA)and glycine receptors (5, 12, 24). Intracellular Cl−may also modifyaspects of cell signaling or ion channel gating (25, 26). Althoughamphotericin B has been reported to produce slightly better andfaster perforation than either nystatin or gramicidin (11, 13), allthree perforants can produce access resistances ~10–20 MW orlower under optimal conditions. Factors beyond the perforant maybe more important for low access recordings, such as appropriatetip-filling times, a steeply tapering pipette shank with low resistance,and a freshly made perforant–pipette solution (11).The need to solubilize the above ionophores in nonaqueoussolvents and dissolve them in pipette solution led to an evaluationof alternate simpler (and less expensive) perforants such as thesaponin derivative b-escin. b-Escin can produce greater and morefrequent perforation than either amphotericin B or nystatin (14, 15),it is more stable in aqueous solution, and it is less costly. However,b-escin pores are significantly larger, with divalent ions and evenhigh-molecular-weight compounds (up to 10–15 kDa) permeating;hence, it is not suitable when native concentrations or small signal-ing molecules need to be maintained. The larger pore size has beenutilized to fill cells with fluorophores such as fluo-2 (14).The following is a step-by-step description of our typical methodfor using amphotericin B or nystatin followed by that forgramicidin. 1. A stock solution of amphotericin B or nystatin is prepared at aconcentration of 50–100 mg/ml in dimethylsulfoxide (DMSO)on the day of the recording. This stock solution is kept awayfrom light and at room temperature. 2. The stock solution (2 ml) is added to 1 ml of internal pipettesolution and mixed using ultrasonication, yielding a finalconcentration of 100–200 mg/ml. The DMSO concentration is0.2% (vol/vol). We avoid filtering these solutions, although Raeet al. (11) reported that amphotericin B may be filtered through0.2-mm filters without loss of activity. Fresh nystatin or amphot-ericin should be added to the pipette solution every 2–3 h.4.3. PracticalGuide toPerforated Patch-Clamp Recordings4.3.1. Amphotericin Bor Nystatin
76 H. Ishibashi et al. 3. Both polyene antibiotics and gramicidin at the tip of the patchpipette may impair the initial GW seal formation; consequently,prefilling the tip of the pipette by brief immersion into an anti-biotic-free solution may be necessary. The optimal time forpipette tip immersion depends on multiple factors, includingpipette shape and diameter [larger tips require less filling time,smaller-tip pipettes (~ > 5 MW) may not need filling], the per-forant concentration used (a lower concentration requires littleor no filling), and the time required between filling and sealing(with slower procedures requiring more tip filling). The timecan be determined empirically, aided by microscopic inspec-tion of tip geometries and filling heights. In our experiencewith filament-free borosilicate glass capillaries of 3–7 MW andwith short and blunt tapers, filling for about 2 s is optimal. Theremainder of the pipette is back-filled with the internal pipettesolution containing the perforant. Small air bubbles at the tipare rapidly and easily removed by tapping the pipette shaft withthe index finger. 4. The patch pipette is secured in the pipette holder and dippedinto the external solution in the recording chamber and guidedtoward the cell as rapidly as possible, before the antibioticdiffuses to the tip. No positive pressure is applied during theapproach. After gently pushing the pipette tip onto the cellmembrane, causing a slight increase of the pipette resistance,gentle negative pressure/suction is applied to obtain the GWseal (Fig. 4.1b). Canceling the fast capacitive transients helpswith subsequent monitoring of perforation. 5. The pipette potential is subsequently typically held between−40 and −70 mV, gradually approaching the cell’s restingmembrane potential as perforation proceeds. The progress ofperforation is monitored by visualizing at high temporal reso-lution the current response to repetitive hyperpolarizingvoltage pulses of about 5–10 mV (DV) (Fig. 4.1b). As the accessresistance (Ra) decreases and electrical contact with the cellimproves, a current transient due to the cell membrane capaci-tance (Cm) is observed. The amplitude of this current transientis given by DV/Ra(hence, the current response increases asperforation proceeds), and the decay time constant is given byCm × Ra(hence, the current decay gets sharper/faster as perfo-ration proceeds). The values of Cmand Racan be quantified byanalyzing the current transient or read from the capacitanceand series resistance cancelation dials off the patch-clampamplifier. Recordings can commence once the Rahas stabilizedat a suitable value and then is compensated for. We routinelyobtain stable Ra < 30 MW by 30 min. Rashould be monitoredand adjusted during the experiment, and data should be usedonly during periods when this value is relatively stable.
774 Perforated Patch ClampThe general procedure for the gramicidin-perforated patch is thesame as for amphotericin or nystatin, with some additional pointsas follows. The stock solution is prepared by dissolving gramicidinD in DMSO at a concentration of 2–50 mg/ml and may be storedfor 1–2 days at −20°C. The patch pipette solution contains grami-cidin at 5–100 mg/ml, with the most appropriate concentrationdetermined in preliminary experiments. Higher concentrationsaccelerate perforation speed but may result in spontaneous rupturingof the patch membrane (this seems to depend on the preparation)and requires prefilling of the pipette tip with gramicidin-free solu-tion. A fresh pipette-filling solution needs to be prepared every1–2 h to obtain low-access resistance recordings.The lipophilic nature of the polyene antibiotics and gramicidinresults in difficulties in using them in physiological solutions,including time-consuming preparation, large solvent concentrations,rapid loss of potency in pipette solutions, and interference with GWseal formation. Some of these problems can be overcome by usingb-escin, but this large pore is not suitable for many applications.Horn and Marty (4) perfused the pipette with nystatin after form-ing the GW seal, but it requires a pipette perfusion system.An alternative method (27, 28) used the bipolar moleculuesfluorescein or N-methyl-d-glucamine (NMDG) to help disperseand solubilize nystatin or amphotericin B. A stock solution is madefrom 5 mg nystatin and 20 mg fluorescein in 1 ml methanol, or froma 0.1 M solution of NMDG dissolved in methanol (pH adjusted toabout 7 with methanesulfonic acid in the presence of 0.01 M phenolred) to which nystatin (5 mg/ml) was added. Immediately beforeuse, 50 ml of the stock solution is placed in a polyethylene test tubeand dried completely with a stream of N2gas. Pipette solution (1 ml)is added to the tube and briefly vortexed. The pipette solution canbe filtered through a 0.22-mm syringe filter; tip filling is not required(hence perforation is achieved more rapidly). Positive pressure canbe applied during the approach to the cell, which makes this particu-larly useful for blind patch-clamping in tissues slices. As with otherperforants, flouroscein is light sensitive. In fact, whole-cell access canbe reversibly closed by the microscope light (27).The activity of some ion channels requires cytoplasmic constituentsthat are lost with the formation of cell-free patches, such as out-side-out patch configuration. Withdrawal of the pipette followingformation of the PPWC configuration can result in formation of aperforated excised vesicle, analogous to an outside-out excisedmembrane patch but in which larger intracellular molecules andhence signaling pathways are maintained (8). The perforated vesicleallows recording of single channels without marked run-down.It also allows investigation of local signal transduction pathwaysthat modulate single-channel activity.4.3.2. GramicidinPerforated Patch4.3.3. SomeAlternatives4.3.4. PerforatedVesicles Preparation
78 H. Ishibashi et al.We illustrate here some of the benefits of PPWC recordings with afew brief examples of responses from acutely isolated and primarycultured neurons.Dialysis of cytoplasm and/or the presence of exogenous Ca2+che-lators (e.g., EGTA) results in a gradual loss of Ca2+from internalstores, particularly when these stores are evoked to release Ca2+.This is illustrated in Fig. 4.2, which shows both conventional andPPWC recordings of K+currents in response to repetitive bathapplication of caffeine, which releases Ca2+from intracellular storesand activates Ca2+-activated K+channels on the cytoplasmic mem-brane (29). The response to caffeine gradually decreases in ampli-tude on conventional whole-cell recordings but is constant forabout ³1 h on the amphotericin B PPWC recordings.A peculiar property of high-voltage-activated Ca2+currents is thatthey show a time-dependent decrease in amplitude during conven-tional whole-cell recordings, a process termed “run-down.” Various4.4. Examplesof the Utility ofPerforated Patch-Clamp Recordings4.4.1. Intracellular Ca2+Homeostasis4.4.2. Run-Down ofVoltage-Activated Ca2+CurrentsFig. 4.2. Perforated patch-clamp whole-cell (PPWC) mode maintains Ca2+stores. Currentresponses to 10 mM caffeine in acutely isolated rat CA1 hippocampal neurons recordedunder conventional whole-cell recordings (a) or amphotericin B PPWC recordings (b) Notethat the response amplitude runs down in (a) but is constant in (b). Caffeine was appliedeach ~5 min. Recordings were performed at a holding potential (VH) of −50 mV.
794 Perforated Patch Clampattempts have been made to prevent run-down, including applicationof substances to maintain channel phosphorylation and/or protectagainst proteolysis, but none of these efforts has been satisfactory.In contrast, run-down of voltage-activated Ca2+channels is mark-edly reduced/delayed using nystatin PPWC recordings (Fig. 4.3).The stable recording of Ca2+channel currents has allowed, forexample, detailed pharmacological dissection of the contributionof different voltage-activated Ca2+channels in different prepara-tions (30, 31).On conventional whole-cell recordings and PPWC recordingsusing the polyene antibiotics or b-escin, the intracellular Cl−con-centration ([Cl−]) equilibrates with that in the pipette [Cl−]. Incontrast, gramicidin PPWC preserves the “normal” intracellular[Cl−] and enables one to record the physiological response ofCl−-permeant channels; it also allows us to investigate the modulationof intracellular Cl−homeostasis. Figure 4.4a compares the responseof ionotropic hippocampal GABA receptors when recorded usinga conventional whole-cell or gramicidin PPWC technique at thesame holding potential (VH) of −50 mV. The direction of the cur-rent response is completely different, reflecting the different intra-cellular [Cl−] and hence driving force. The physiological range ofintracellular [Cl−] (~5–30 mM) is less than often used in pipettesolutions in conventional whole-cell recordings (~150 mM). Thegramicidin PPWC technique can be used to measure the intracel-lular [Cl−] as shown in Fig. 4.4b. Currents through the anion-selective ionotropic GABA or glycine receptors are measured atdifferent holding potentials, and a current–voltage curve can be4.4.3. GABA andGlycine ResponsesRecorded by theGramicidin-PerforatedPatch RecordingFig. 4.3. Nystatin PPWC mode prevents the run-down of Ca2+channel currents. Typical traces of high-voltage activated Ca2+currents recorded from acutely isolated rat intracardiac ganglion cells using conventional (left panel) or PPWC (right panel)recordings. Current traces obtained at the beginning of the experiment (control) and 30 min later (30 min) are superimposed.
Fig. 4.4. Physiological g-aminobutyric acid (GABA) and glycine responses and intracellular[Cl−] measurements using the gramicidin PPWC technique. (a) GABA-induced outwardand inward currents recorded at a VHof −50 mV before (left trace) and after (right trace)rupture of the membrane during a gramicidin PPWC recording. Membrane rupture resultsin a much higher intracellular [Cl−] as the cell equilibrates with the pipette [Cl−] (~150 mM).Hence, the GABA response changes from an outward current (representing Cl−efflux) toan inward current (Cl−influx). (b) Glycine-induced currents recorded using the gramicidinPPWC recording at various VHs values in cultured spinal cord neurons are measured toconstruct a corresponding current–voltage curve from which the Vrev(arrow) is deter-mined. The intracellular [Cl−] is estimated from a derivation of the Nernst equation:[Cl−]in = [Cl−]outexp(VrevF/RT), where F is Faraday’s constant (96,485 Cmol−1), R is the gasconstant (8.3145 VCmol−1 K−1), and T is absolute temperature (293.15 K at 20°C). (c) Suchmeasurements are used to demonstrate that furosemide causes a reversible increase inthe intracellular [Cl−]. This is due to direct inhibition of K+-coupled Cl−efflux via the neu-ronal KCC2 transporter. The graph plots the mean ± SEM from five experiments.
814 Perforated Patch Clampplotted. If using ramp voltage responses, control currents in theabsence of GABA or glycine should be subtracted from those inthe presence of GABA or glycine. The X axis intercept of this curve(the reversal potential, Vrev) gives the equilibrium potential wherethe driving force due to the membrane voltage cancels out thatfrom the concentration gradient. This is expressed mathematicallyby the Nernst equation, from which the intracellular [Cl−] can beestimated (Fig. 4.4b). Precise quantification of Vrevand intracellu-lar [Cl−] requires consideration of all permeant ion species, activitycoefficients, and liquid junction potentials (32); the latter can beup to ~10 mV or more if using larger ions in the pipette solution.These recordings should use a K+-based solution in the pipette, asthe intracellular [Cl−] is sensitive to transmembrane K+(and, to alesser extent, Na+) gradients, and some other cations (e.g., Cs+) cansignificantly inhibit some of the key Cl−transporters (33).Figure 4.4c shows the effect of furosemide, a Cl−transporter inhib-itor, on the intracellular [Cl−] in isolated hippocampal neurons.The ability to measure the intracellular [Cl−] by the gramicidinPPWC technique has facilitated revealing developmental- andinjury-induced changes in intracellular Cl−homeostasis (34, 35).This chapter has highlighted some of the benefits of the PPWCtechnique, but there are also some drawbacks potential users needto consider. First, PPWC recordings are much more time-consumingthan conventional whole-cell recordings: fresh perforant-containingpipette solutions are required, and there is a wait (often ³30 min)for perforation and stabilization to occur. Second, only under themost optimal conditions can access/series resistances similar tothose under conventional whole-cell recordings be obtained.Usually they are much higher and one must (as with conventionalwhole-cell recordings) be aware of the voltage errors and filteringeffects of this access resistance. A voltage offset (=pipette current ×Ra) must be subtracted from VH, and the −3 dB of the filteringeffect = 1/[2p × Ra × Cm]. Series resistance compensation can reducethese errors. Finally, one must also be aware of a Donnan potentialbetween the VHand the real membrane potential that arises as thelarge anions in the cell cannot equilibrate with the patch pipettesolution. As discussed by Horn and Marty (4), this may be as highas ~10 mV with a KCl pipette solution. This potential can bereduced by including large impermeant ions in the pipette, but it isdifficult to measure or estimate it precisely. Hence in PPWC recordings,one must be aware of potential inaccuracies in citing absolutevoltages associated with current responses (e.g., Kdfor activationor inactivation).4.5. Drawbacksof the PerforatedPatch-ClampTechnique
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