[14] Purification and functional reconstitution of high-conductance calcium-activated potassium channel from smooth muscle

274

PURIFICATION AND RECONSTITUTION

[ 14]

[14] P u r i f i c a t i o n a n d F u n c t i o n a l R e c o n s t i t u t i o n of High-Conductance Calcium-Activated Potassium Channel from Smooth Muscle

By

MARIA

L. G A R C I A , K A T H L E E N M. G I A N G I A C O M O , M A R K U S H A N N E R , HANs-GONTHER KNAUS, OWEN B. MCMANUS,

W I L L I A M A . SCHMALHOFER, and G R E G O R Y J. KACZOROWSKI

Introduction Potassium channels constitute the largest and most diverse family of ion channels. 1 These channels can be categorized according to their biophysical and pharmacologic properties, and all share in common a high selectivity for K + as the permeant ion. As a first approximation, K + channels can be divided into either voltage-gated or ligand-gated channels, depending on the stimulus that triggers conformational changes leading to channel opening. High-conductance Ca2+-activated K + (maxi-K +) channels are activated by both membrane depolarization and binding of Ca 2+ to sites at the intracellular face of the channel. Maxi-K + channels are present in both electrically excitable and nonexcitable cells, and display high conductance and selectivity for K +. These channels are involved in regulation of the excitationcontraction coupling process in smooth muscle, as well as in control of transmitter release from neuroendocrine tissues. The pharmacology of maxi-K + channels has been developed during the last few years and efforts are continuing to identify novel and selective modulators of this channel family. 2,3 Development of the molecular pharmacology of maxi-K + channels was greatly facilitated by discovery of charybdotoxin (ChTX), a peptidyl channel inhibitor that is a minor component of Leiurus quinquestriatus vat. hebraeus venom. 4 ChTX binds in the outer vestibule of maxi-K + channels with a 1 : 1 stoichiometry and blocks ion conduction by physically occluding the channel pore. Purification and subsequent radiolabeling of ChTX with Na125I to high-specific activity provided a tool with which to identify maxi-K + chan1 A. Wei, T. Jegla, and L. Salkoff, Neuropharmacology 35, 805 (1996). z M. L. Garcia, M. Hanner, H.-G. Knaus, R. Koch, W. Schmalhofer, R. S. Slaughter, and G. J. Kaczorowski, Adv. Pharmacol. 39, 425 (1997). 3 G. J. Kaczorowski, H.-G. Knaus, R. J. Leonard, O. B. McManus, and M. L. Garcia, J. Biomembr. Bioenerg. 28, 255 (1996). 4 M. L. Garcia, H.-G. Knaus, P. Munujos, R. S. Slaughter, and G. J. Kaczorowski, Am. J. Physiol. 269, C1 (1995).

METHODS 1N ENZYMOLOGY.VOL. 294

Copyright © 1999by AcademicPress All rights of reproductionin any form reserved. 0076-6879/99 $30.00

[14]

PURIFICATION AND RECONSTITUTIONOF maxi-K CHANNEL

275

nels in native tissues and to develop their molecular pharmacology. Another important question concerns the exact molecular composition of native maxi-K ÷ channels. We know that some properties of the channel, such as Ca 2+ sensitivity, vary greatly from tissue to tissue. This could be explained by either the presence of distinct pore-forming subunits that would possess different sensitivities to Ca 2+, or by modulation of the pore-forming subunit by other auxiliary subunits that are closely associated with the pore. This, in fact, is the situation found for Na + and Ca 2+ channels; these channels exist in v i v o as multiple-subunit complexes, where auxiliary proteins have major effects on properties of the subunit that constitutes the pore. To date many of the auxiliary subunits of ion channels have been identified after purification of the channel preparation to homogeneity from native tissues. In fact, it would be difficult to achieve the same results using molecular biological approaches (e.g., expression-cloning techniques) because there is no way to predict which parameter of the channel would be altered by coupling to various auxiliary subunits. Thus, purification of a native channel complex remains a viable approach by which to determine subunit composition of a channel. In this article, we discuss procedures that have been developed to purify maxi-K + channels from smooth muscle tissues, as well as how to reconstitute the purified preparation into liposomes for determination of channel activity. In addition, we also discuss ways of obtaining protein sequence information from components of the maxi-K + channel preparation that are useful for obtaining full-length c D N A clones of these proteins. Solubilization of Maxi-K + C h a n n e l s To achieve a functionally active, homogeneous maxi-K ÷ channel preparation, three parameters must be established. First, a marker for the channel must be identified that can be used to track the protein during purification. For this purpose, we used 125I-labeled ChTX binding to maxi-K + channels. 5 Monitoring the effect of other channel modulators on the binding reaction provides a way of assessing the integrity of the channel preparation. Second, it is necessary to identify a source of tissue that possesses significant amounts of the target. Out of the sources that we screened for maxi-K + channels (i.e., neuronal, skeletal muscle, and smooth muscle tissues), we found that bovine tracheal and aortic smooth muscle displayed the highest density of ~25I-labeled ChTX binding sites. Finally, the detergent used for extraction of the channel from its lipid environment must maintain the structural 5j. Vazquez, P. Feigenbaum, V. F. King, G. J. Kaczorowski,and M. L. Garcia, J. Biol. Chem. 265, 15564 (1990).

276

PURIFICATION AND RECONSTITUTION

[ 14]

integrity of the protein in its solubilized form, and the channel complex must remain stable for the extended period of time that is required to carry out all purification steps. Out of all detergents that we tested, only CHAPS and digitonin yielded both channel solubilization and initial toxin binding activity. 6 However, activity was found to be more stable in the presence of digitonin; therefore, this was the detergent of choice for attempting maxiK + channel purification. Purified sarcolemmal membrane vesicles derived from either bovine tracheal or aortic smooth muscle are prepared by methods that are well described in the literature. 7,s These membranes can be stored at - 7 0 ° until use without loss of [125I]ChTX binding activity. Before solubilization, membranes are thawed and the following protease inhibitors added: 1 m M iodoacetamide, 0.1 m M phenylmethylsulfonyl fluoride, and 0.1 m M benzamidine (these agents are present throughout the entire purification procedure). Digitonin is then added from a 10% (w/v) stock solution to a final concentration of 0.5%, and the mixture is incubated at 4 ° for 10 min with continuous shaking. It is worth noting that digitonin, being a natural product, can vary significantly in its physical properties between different lots and manufacturers. We have found that digitonin special grade (watersoluble), purchased from Biosynth A G (Skokie, IL), gives highly reproducible results. The easiest way to prepare a 10% (w/v) solution of digitonin is to sonicate the suspension for a few minutes. Once in solution, the material is filtered through 0.2-/xm cellulose acetate filters to eliminate any particulate. At the end of the 10-min exposure to digitonin, the membrane suspension is subjected to centrifugation for 50 min at 180,000g at 4 °. The supernatant ($1) contains large amounts of contaminant proteins, but no significant [125I]ChTX binding activity, and can therefore be discarded. The pellet is homogenized using a glass-Teflon homogenizer in 20 m M NaC1, 20 m M Tris-HC1, p H 7.4; digitonin is added to a final concentration of 1%; and the suspension is incubated at 4 ° as indicated earlier. After centrifugation to separate soluble from particulate material, the supernatant ($1) is retained and the pellet is subjected to another extraction with detergent as previously indicated. We have found that for optimal recovery of solubilized [125I]ChTX binding sites, the extraction procedure needs to be repeated up to a total of six times. The supernatants from the second to the sixth extraction ($2_6) are combined for further processing. Solubilized material 6 M. Garcia-Calvo,J. Vazquez, M. Smith, G. J. Kaczorowski,and M. L. Garcia, Biochemistry 30, 11157 (1991). 7R. S. Slaughter,J. L. ShevelLJ. P. Felix, M. L. Garcia, and G. J. Kaczorowski,Biochemistry 28, 3995 (1989). s R. S. Slaughter, A. F. Welton, and D. W. Morgan, Biochim. Biophys. Acta 904, 92 (1987).

[14]

PURIFICATION AND RECONSTITUT1ON OF

maxi-K

277

CHANNEL

TABLE I PURIFICATION OF CHTX RECEPTOR FROM BOVINE TRACHEAL SMOOTH MUSCLEa 1251-ChTX

Specific

Binding Step Membranes Sz 6 DEAE-Sepharose WGA-Sepharose Mono Q

Hydroxylapatite First sucrose gradient Mono S

Second sucrose gradient

pmol

%

8014 4268 100 2712 63.5 2292 53.7 1627 38.1 1138 26.6 801 18.7 248 5.8 142 3.3 42 b 1.Oh

Protein mg

%

10067 6327 100 1762 27.8 453 7.1 81 1.3 8.5 0.13 2.6 0.04 0.38 0.006 0.13 0.002 0.032 h 0.0005 b

activity (pmol/mg

Purification

protein)

(x-fold)

0.79 0.67 1.54 5.05 20.08 133.88 308.07 652.63 1092.30 1312.50 b

1.0 2.3 7.5 30.0 199.8 459.8 974 1630 1959 b

" Amounts referred to starting material derived from --250 cow tracheas. [Reprinted with permission from the American Society of Biochemistry and Molecular Biology, Inc., from M. Garcia-Calvo et al. J. Biol. Chem. 269, 676 (1994).] b Highest specific activity fraction from the gradient.

contains as much as 50-60% of the binding sites present originally in membranes, with a similar specific activity as defined by pmol [125I]ChTX binding sites/mg protein (Table I). 9'l° Determination of [125I]ChTX binding to solubilized receptors can be easily accomplished by filtration techniques. 6 For this purpose, solubilized material in 0.05% (w/v) digitonin is incubated with [125I]ChTX in a medium consisting of 10 mM NaC1, 20 mM Tris-HC1, pH 7.4, 0.1% (w/v) bovine serum albumin (BSA), at room temperature until equilibrium conditions are achieved (ca. 1 hr). At the end of the incubation period, 10/xl of a 50 mg/ml (w/v) y-globulin solution is added, followed by addition of 4 ml of 10% (w/v) polyethylene glycol ( M r ~ 8 0 0 0 ) in 100 mM NaC1, 20 mM TrisHC1, pH 7.4. The precipitate is immediately collected onto GF/C glass fiber filters that have been presoaked in 0.5% polyethyleneimine, and filters are rinsed twice with 4 ml of polyethylene glycol quench buffer. For determination of nonspecific binding, parallel incubations are carried out in the presence of 10 nM ChTX. M. Garcia-Calvo, H.-G. Knaus, O. B. McManus, K. M. Giangiacomo, G. J. Kaczorowski, and M. L. Garcia, J. Biol. Chem. 269, 676 (1994). ~o K. M. Giangiacomo, M. Garcia-Calvo, H.-G. Knaus, T. J. Mullmann, M. L. Garcia, and O. McManus, Biochemistry 34, 15849 (1995).

278

PURIFICATION AND RECONSTITUTION

[ 141

DEAE-Sepharose Chromatography To facilitate the following purification steps, solubilized ChTX receptor is adjusted to 50 mM NaC1, and loaded onto a DEAE-Sepharose CL-6B column equilibrated with 50 mM NaC1, 20 mM Tris-HC1, pH 7.4, 0.1% digitonin. After washing the resin thoroughly with equilibration buffer, bound receptor is eluted batchwise with 170 mM NaC1, 20 mM Tris-HC1, pH 7.4, 0.1% digitonin. This step allows for >60% recovery of solubilized receptors with more than 2-fold enrichment in [125I]ChTX binding activity (Table I). Importantly, a large amount of protein is removed at this step which otherwise would cause a significant decrease in the yield at the next purification step. Wheat Germ Agglutinin--Sepharose Chromatography The DEAE-Sepharose eluted receptor is incubated overnight at 4 ° with wheat germ agglutinin (WGA)-Sepharose in 200 mM NaC1, 20 mM TrisHC1, pH 7.4, 0.1% digitonin. The suspension is then placed in an empty column, and the fluid phase is collected until the WGA-Sepharose resin is packed. The column is washed with 10 bed volumes of equilibration buffer to remove unbound material. Glycoproteins are then eluted with 200 mM N-acetyl-D-glucosamine present in the equilibration buffer. The eluate is dialyzed against 20 mM Tris-HC1, pH 7.4, 0.05% digitonin, then concentrated 20-fold by use of an Amicon (Danvers, MA) ultrafiltration cell, and finally adjusted with NaC1 to a final concentration of 200 mM. This step allows for recovery of >50% of solubilized ChTX receptors, with >7-fold enrichment in specific activity (Table I). It is important to take care that sufficient amounts of WGA-Sepharose resin be employed to provide for full retention of glycoproteins. Otherwise significant quantities of material will appear in the eluate and this will have to be processed again using more resin. For this reason, the DEAE-Sepharose step is critical in order to remove contaminating material that will decrease the loading capacity of the WGA-Sepharose resin. Mono Q Ion-Exchange Chromatography Mono Q ion-exchange chromatography, although yielding modest enrichment of the ChTX receptor, is crucial in that it allows the subsequent step in purification to proceed optimally. We employ a Mono Q H R 10/ 10 (Pharmacia, Piscataway, NJ) ion-exchange column equilibrated with 100 mM NaC1, 20 mM Tris-HC1, pH 7.4, 0.05% digitonin, which is operated at room temperature. Because loading capacity of the column is limited to

[14]

P U R I F I C A T I O N A N D R E C O N S T I T U T I O N OF m a x i - K

CHANNEL

279

ca. 100 mg of protein, caution should be used so as not to overload it. Thus, the WGA-Sepharose eluted material, after being filtered through 0.2-~m filters, is usually applied in several consecutive runs. Elution of bound material is achieved with a linear gradient of NaC1 (0.1-0.5 M) over 70 min at a flow rate of 2 ml/min; 4-ml fractions are usually collected. Fractions containing higher specific activity of ChTX binding elute between 0.21 and 0.31 M NaC1. This ion-exchange chromatography yields ca. 40% recovery of solubilized ChTX receptors, with a 30-fold enrichment in specific activity (Table I).

Hydroxylapatite Chromatography Fractions from the Mono Q ion-exchange column are adjusted to 80 mM sodium phosphate, pH 7.0, filtered through 0.2-~m filters, and loaded onto a Bio-Gel HPHT (Bio-Rad, Richmond, CA) 100 x 7.8-mm hydroxylapatite column equilibrated with 80 mM sodium phosphate, pH 7.0, 10 mM NaC1, 0.05% digitonin, at room temperature. Because the loading capacity of this column is limited to ca. 25 mg, it is necessary to be cautious with the amount of material applied. If necessary, the Mono Q eluate should be loaded in several consecutive runs. Bound material is eluted with a linear gradient of 80-160 mM sodium phosphate in 10 mM NaC1 applied within 12 min, followed by a linear gradient from 160 mM sodium phosphate in 10 mM NaC1 to 560 mM sodium phosphate, 70 mM NaC1 applied within 10 min, at a flow rate of 0.5 ml/min. Fractions of 1 ml are collected, and those containing the highest specific activity of ChTX binding, elute between 200 and 440 mM sodium phosphate. These fractions contain ->25% of the solubilized receptors and are enriched ca. 200-fold (Table I).

Sucrose Density Gradient Centrifugation Fractions from the hydroxylapatite column are dialyzed against 20 mM Tris-HC1, pH 7.4, 0.05% digitonin, and concentrated using an Amicon ultrafiltration cell. The material is then applied to a continuous 7-25% (w/v) sucrose gradient in 20 mM Tris-HC1, pH 7.4, 0.05% digitonin, separated by centrifugation for 12 hr at 34,000 rpm in a Beckman SW 40 Ti rotor (Palo Alto, CA), and fractionated into 0.6-ml fractions. ChTX binding activity migrates as a large particle with an apparent sedimentation coefficient of 23S. The recovery of solubilized ChTX receptors is ca. 20% with >450fold enrichment in specific activity (Table I). Mono S Ion-Exchange Chromatography Active fractions from the sucrose density gradient centrifugation step are dialyzed against 20 mM 2[N-morpholino]ethanesulfonic acid (MES-

280

PURIFICATION AND RECONSTITUTION

[141

NaOH), pH 6.2, 0.05% digitonin, and loaded onto a Mono S HR 5/5 (Pharmacia, Piscataway, N J) ion-exchange column that had been equilibrated with the same buffer at room temperature. Bound material is eluted at a flow rate of 0.5 ml/min with a linear gradient of NaCl (0-700 mM in 20 min). ChTX binding activity elutes between 120 and 260 mM NaC1. Recovery of solubilized receptors is ca. 6% with almost 1000-fold enrichment in specific activity (Table I). Sucrose Density Gradient Centrifugation A density gradient approach was again chosen for the last purification step because it takes advantage of the large mass of the ChTX receptor, thus allowing easy separation from other remaining small molecular weight contaminating protein components. Before applying to a continuous 7-25% (w/v) sucrose gradient, fractions from the Mono S ion-exchange column are dialyzed against 20 mM Tris-HC1, pH 7.4, 0.01% digitonin, and the sample is concentrated. Protein components are separated as indicated before. The average recovery of receptors is ca. >3% with >1600-fold enrichment (Table I). The fraction with the highest specific activity is enriched ca. 2000-fold with respect to starting material and represents 1% of initial binding activity. The specific activity of the final preparations, 1.3 nmol [125I]ChTX binding sites/mg protein, is very close to the value estimated for a pure preparation consisting of a tetrameric structure of four pore forming subunits and four auxiliary subunits (see below). The recovery of activity at each step of purification approaches 100%. This implies that no substantial loss of activity occurs during the time involved in the purification procedure. This is remarkable since, depending on the amount of starting material used, the whole procedure can take several days. For instance, purification that involves material derived from 250 cow tracheas (Table I) can take up to 10-14 days to be completed. Proteolytic degradation of the pore-forming subunit, however, occurs during the hydroxylapatite chromatography step. This has been noted in immunoblot experiments employing site-directed antibodies raised against different peptide sequences of the protein. Although initially all antibodies recognize a single polypeptide in membrane preparation with the expected Mr (125,000), and continue to do so throughout the first steps in purification, after the hydroxylapatite column, the pattern of recognition changes due to specific cleavages in the C-terminal region of the protein, u These cleavages, however, do not cause any loss in [125I]ChTX binding activity and, since the 11H.-G. Knaus,A. Eberhart, R. O. A. Koch,P. Munujos,W. A. Schmalhofer,J. W. Warmke, G. J. Kaczorowski,and M. L. Garcia,J. BioL Chem. 270722434 (1995).

[14]

PURIFICATION AND RECONSTITUTION OF m a x i - K CHANNEL

281

mass of the receptor remains unchanged, we believe that the fragments remain associated through disulfide bridges. Evidence supporting this hypothesis comes from the fact that if the final preparation is subjected to SDS-PAGE in the absence of reducing agents, all antibodies appear to recognize a single polypeptide corresponding to the full-length protein. We do not know the exact nature of the proteolytic cleavage that takes place at that specific stage in purification, but we speculate that it may be due to some conformational change that occurs in the protein on binding to the hydroxylapatite matrix followed by the action of a protease that copurities with the receptor. One can also speculate that the proteolytic cleavage is self-inflicted. The carboxy terminus of the o~subunit of the bovine smooth muscle maxi-K+ channel contains a domain homologous with serine proteases, although sequence analysis suggests that this domain is probably inactive. 12 The purified ChTX receptor consists of two subunits, the pore-forming subunit (o0 and an auxiliary subunit (/3). Detection of the/3 subunit by silver staining after SDS-PAGE is virtually impossible. However, the /3 subunit can be visualized by either of two independent methods. The first involves covalent incorporation of [125I]ChTX into the /3 subunit in the presence of a bifunctional cross-linking reagent. As previously demonstrated with intact membranes, incorporation of radioactivity takes place exclusively into a polypeptide with a Mr of 3 5 , 0 0 0 . 6'9'1° On deglycosylation, a final product of 25,600 is obtained, which corresponds to the size of the core protein, 21,200, given that 4400 of this mass is contributed by the radiolabeled toxin. It is also possible to detect the/3 subunit after labeling the purified preparation with 125I-labeled Bolton-Hunter reagent. The/3 subunit displays an identical time course of deglycosylation as that of the [125I]ChTX cross-linked protein, and the apparent molecular weight of the deglycosylated ~25I-labeled Bolton-Hunter labeled core protein is 21,400. 9 Interestingly, the electrophoretic mobility of the o~ subunit is not altered after N-glycanase treatment, indicating that this protein is not glycosylated by N-linked sugars. These findings are in agreement with the postulated transmembrane topology of this protein, in which putative N-linked glycosylation sites are placed on the inner face of the membrane. 13 The pharmacologic properties of the purified ChTX receptor are identical to those characteristic of the receptor in intact membranes. 9,1° Thus, not only does the affinity of [125I]ChTX remain unchanged, but the ability of other agents to modulate the binding reaction is also unchanged. These agents include other related peptidyl inhibitors of maxi-K + channels, such 12 G. W. J. Moss, J. Marshall, and E. Moczydlowski, J. Gen. Physiol. 108, 473 (1996). 13 M. Wallner, P. Meera, and L. Toro, Proc. Nat. Acad. Sci. U.S.A. 93, 14922 (1996).

282

PURIFICATION AND RECONSTITUTION

[14]

as iberiotoxin and limbatustoxin, as well as small organic molecules such as tetraethylammonium ion, all of which bind to the outer vestibule of the channel. In addition, ions such as potassium, barium, and cesium, which bind with high affinity to sites located along the ion conduction pathway, display identical Ki values in the purified preparation as those found with intact membranes. Finally, members of the indole diterpene family of maxiK + channel inhibitors modulate [125I]ChTX binding to the purified receptor in the same fashion as they do in native preparations. For instance, paxilline causes a concentration-dependent stimulation of toxin binding, whereas aflatrem produces full inhibition of the binding reaction. In summary, the procedure described above yields a homogeneous preparation consisting of two subunits that displays all the pharmacologic properties of the native ChTX receptor in smooth muscle. Reconstitution of ChTX Receptor into Liposomes To demonstrate that the purified ChTX receptor is indeed the maxiK + channel, it is necessary to reconstitute the preparation in a system appropriate for making electrical recordings of single-channel activity. The biophysical arid pharmacologic properties of native maxi-K + channels have been well characterized after incorporation of channels from membrane vesicle preparations into artificial phospholipid bilayers. However, the presence of digitonin in the purified preparation precludes such recordings due to the instability of the bilayers formed. Therefore, we elected to reconstitute the purified preparation into liposornes first in order to eliminate detergent, and then fuse the liposomes with an artificial lipid bilayer. Because the critical micellar concentration (CMC) of digitorlin is very low, it is necessary to break up the micelles with a second detergent, such as 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid (CHAPS). For this purpose, aliquots of purified receptor in 0.05% (w/v) digitonin are incubated on ice with 0.3% BSA, 0.34% L-a-phosphatidylcholine, and 0.85% CHAPS for 30 min. We have found that the amount of extract in 0.05% digitonin to achieve optimal reconstitution is critical, and that 300/xl of material yields good results. The mixture is then applied to a 1-rnl Extracti-Gel D column (Pierce, Rockford, IL) equilibrated with 100 mM NaC1, 20 mM HEPES-NaOH, pH 7.4, 0.2% BSA, and eluted with 1.5 ml of 100 mM NaC1, 10 rnM MgC12, 20 mM HEPES-NaOH, pH 7.4. Proteoliposomes are then precipitated by addition of polyethyleneglycol (Mr -- 8000) to give a final concentration of 25%, and collected by centrifugation at 100,000 rpm (Beckman TLA 100.3 rotor) for 20 min. Proteoliposomes are washed once in 100 mM NaCI, 20 mM HEPES-NaOH, pH 7.4, collected by centrifugation as indicated above, resuspended in washing medium, frozen in liquid N2, and stored at -70 °. There is no loss of biologi-

[14]

P U R I F I C A T I O N A N D R E C O N S T I T U T I O N OF

maxi-K

CHANNEL

283

cal activity after storage under these conditions for up to several months. The efficiency of reconstitution varies from 25 to 60% as determined by [125I]ChTX binding to the proteoliposome preparation. In the absence of digitonin, toxin binding is about 30% of the amount observed in the presence of this detergent, suggesting that the receptor preferentially reconstitutes in the inside-out orientation. 9 Fusion of Reconstituted ChTX Receptors into Lipid Bilayers Proteoliposomes were fused with lipid bilayers composed of either neutral zwitterionic lipids [1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmiioyl-2-oleoylphosphatidylcholine(POPC) in a 7/3 molar ratio] or a combination of neutral and charged lipids [POPE and 1palmitoyl-2-oleoylphosphatidylserine (POPS) (Avanti Polar Lipids, Birmingham, AL) in a 1/1 molar ratio]. The lipids were dissolved in decane (50 mg/ml) and applied to a 250-/xm hole in a polycarbonate chamber. The use of charged lipids facilitated fusion of proteoliposomes with the bilayer, but neutral lipids were used in experiments where it was desirable to minimize the effects of lipid surface charge on channel function. The hole in the dry polycarbonate cup was pretreated by application of a small amount of lipid solution that was allowed to dry before adding the aqueous solutions. This step enhanced the stability of the bilayers, which would typically last for many hours. Bilayers were formed by painting a very small volume of lipid solution over the hole and monitoring membrane capacitance. Proteliposome fusion with the bilayer was enhanced by establishing an osmotic gradient across the bilayer. Typically, the proteoliposomes were added to the cis side containing 150 mM KC1, 10 mM HEPES and 10 ~M CaC12, pH 7.20, with KOH. The opposite, trans, side contained 10-25 mM KC1, 10 mM HEPES, and 10/xM CaC12, pH 7.20, with KOH. Using higher KC1 concentrations of up to 1 M on the cis side enhanced the rate of vesicle fusion. Direct application of small amounts of the proteoliposome preparation to the bilayer by a glass rod or a microliter pipette is an efficient use of the purified preparation. After channel insertion into the bilayer, the osmotic gradient was collapsed by addition of a small volume of a concentrated KC1 solution (2 M KC1, 10 mM HEPES, pH 7.20) to the trans side, which reduced the probability of further channel insertion. Channels inserted with either polarity, which was determined from the voltage and calcium dependence of channel open probability. Membrane currents were recorded using commercial voltage-clamp amplifiers (Dagan 3900 and List EPC7, Minneapolis, MN). Patch-clamp amplifiers with relatively large input capacitances (especially those with integrating headstages) worked best because they are stable when connected with a large bilayer source capacitance and provide relatively constant frequency

284

PURIFICATION AND RECONSTITUTION

[14]

response in the face of experiment-to-experiment variations in bilayer capacitance. The amplifier was connected to small wells filled with 0.2 M KC1 by silver electrodes coated with silver chloride. These small wells were connected to the bilayer chambers by agar brides containing 0.2 M KC1. Properties of R e c o n s t i t u t e d C h a n n e l s After fusing proteoliposomes containing ChTX receptors purified from bovine trachea and aorta with the bilayer, we routinely observed highconductance channels with the biophysical and pharmacologic properties of smooth muscle maxi-K + channels. Single-channel conductance was about 250 pS in neutral lipids and 320 pS in charged lipids. The single-channel conductance of the channel purified from aorta measured in neutral lipids was identical with the conductance of native channels from aorta. The conductance of the purified channels was highly selective for potassium over sodium or chloride and increased as the potassium concentration was raised. Perhaps the most defining characteristic of maxi-K + channels is their dual regulation by calcium and membrane potential. Open probability of channels purified from either aorta or trachea increased with membrane depolarization with an e-fold increase in open probability per 10-12 mV depolarization. Internal calcium shifted this voltage activation curve to the left as expected for native maxi-K + channels. At a constant membrane potential, calcium increased channel open probability with a Hill coefficient of 2.9 for aortic channels suggesting that three to four or more calcium ions can bind during maximal activation. The distributions of channel open and closed times were described by three and five to six exponential components, respectively, suggesting three open and five to six shut channel states. These kinetic properties are similar to what has been reported for maxiK + channels in other tissues 14 and are precisely what is expected for a channel activated by binding of four or more calcium ions. The purified channel showed transitions between discrete gating modes, suggesting that moding behavior is intrinsic to the channel complex. The pharmacologic properties of the reconstituted channels were as expected for maxi-K + channels from smooth muscle. Externally applied tetraethylammonium ( T E A ) caused a rapid block of the channels that resulted in a time-averaged reduction in single-channel conductance. The Ki for block at 0 mV was 193 /xM and the block increased at negative membrane potentials. The voltage dependence of block was described by a simple model where T E A bound to a site located in the pore and the ~40. B. McManus, J. Bioenerg. Biomembr. 23, 537 (1991).

[14]

PURIFICATION AND RECONSTITUTION OF

maxi-K

CHANNEL

285

bound particle sensed 19% of the membrane field. ChTX blocked the channels by a bimolecular mechanism with a Kd value of 4.6 nM. The mean toxin-blocked time of 30 sec was briefer than the value of 64 sec previously observed for native channels from bovine aorta. 15 The channel behavior of the reconstituted ChTX closely resembles the properties of maxi-K + channels in native smooth muscle and suggests that the purified complex is sufficient to reconstitute most aspects of channel function. Isolation of S u b u n i t s a n d Amino Acid S e q u e n c e Analysis One of the major goals of purifying a native channel is to determine its subunit composition, and obtain partial amino acid sequence from the subunits so that these data can be used to isolate full-length cDNAs. Many methods have been described in the literature for obtaining amino acid sequences from purified proteins. We elected to separate the maxi-K + channel subunits by S D S - P A G E , electroelute the proteins from the gel, subject them to proteolytic digestion, and purify the fragments by microbore high-performance liquid chromatography (HPLC). For the /3 subunit, fractions from the final sucrose density gradient centrifugation containing - 3 1 pmol of [125I]ChTX binding sites were dialyzed against 10 m M sodium borate, p H 8.8, 0.05% Triton X-100 and then reacted with 50/~Ci of 125I-labeled B o l t o n - H u n t e r reagent for 15 rain on ice. The iodinated sample was separated by S D S - P A G E on 12% gels, and the wet gel was exposed for 30 rain to Kodak (Rochester, NY) X A R film to localize the position of the proteins. The area of radioactivity corresponding to the/3 subunit was cut from the gel and electroeluted for 12 hr in 0.1 M ammonium acetate, 0.1% SDS. The sample was then dialyzed against 40 mM sodium phosphate, pH 7.8, 0.02% SDS for 24 hr, concentrated to 50/M, and incubated with 5 /~g of V8 endoproteinase Glu-C for 14 hr at room temperature. The digestion mixture was then loaded onto a Vydac column (Hesperia, CA) (RP-300, 5 /~m, 150 x 2.1 ram) that had been equilibrated with 2% acetonitrite, 10 mM trifluoroacetic acid, using an ABI 130A separation system (Foster City, CA). Elution of bound material was achieved in the presence of a linear gradient of 2-99% acetonitrile at a flow rate of 50/zl/min, and peaks were collected manually. The separated peptides were loaded onto Porton peptide filter supports and subjected to automated Edman degradation employing an integrated microsequencing system (Porton Instruments P12090E, Fullerton, CA) with an on-line detection system. Using this approach, we were able to obtain a 28-amino-acid 15K. M. Giangiacomo, E, E. Sugg, M. Garcia-Calvo, R. J. Leonard, O. B. McManus, G. J. Kaczorowski, and M. L. Garcia, Biochemistry 32, 2363 (1993).

286

PURIFICATION AND RECONSTITUTION

[14]

sequence from one of the peptides that enabled us to construct oligonucleotide probes to screen c D N A libraries and isolate the full-length c D N A coding for the/3 subunit of the maxi-K + channel. 16 To obtain internal amino acid sequence from the o~subunit, the purified maxi-K + channel was subjected to S D S - P A G E and the area of the gel corresponding to the a subunit was cut out, and electroeluted as previously described. After electroelution, samples were reduced in the presence of 4% SDS with 1% 2-mercaptoethanol, alkylated with iodoacetic acid, and dialyzed for 18 hr against 6 M urea, 10 m M sodium phosphate, p H 7.2, containing 20g/liter Dowex A G 1 x 2 resin. Dialysis continued for 24 hr against 5 m M Tris-HC1, p H 8.5, 0.05% CHAPS. Samples were then concentrated 20-fold and incubated with 4/xg of trypsin (final concentration of 80/xg/ml) for 15 hr at 37 °. The digested o~ subunit was then loaded onto an Applied Biosystems Aquapore C18 column (Foster City, CA) (RP-300, 7 ~m, 100 x 1 mm), equilibrated with 2% acetonitrile, 7 m M trifluoroacetic acid, at a flow rate of 30 tzl/min. Elution was achieved in the presence of a linear gradient from 2 to 98% acetonitrile (0.66%/min), and peaks were collected manually. The separated peptides were loaded onto Porton peptide filter supports and subjected to automated Edman degradation. Most of the peaks did not yield any sequence, but we were able to determine unambiguous amino acid sequence from seven of them. 17 All of the sequences can be aligned in very high homology or even identity with the deduced amino acid sequence of Slo, a maxi-K ÷ channel protein cloned from different sources and tissues. These data indicate that the o~ subunit of the purified maxi-K channel from smooth muscle is a member of the Slo family of K + channels. Conclusion The ion channel superfamily, and in particular the K + channel family, continues to grow. From cloning of the Caenorhabditis elegans genome, it is estimated that perhaps 50-80 potassium channel genes exist in that organism. I From the sequence data accumulated so far, eight families of potassium channel genes are conserved between C. elegans and vertebrate species. However, the existence of auxiliary subunits of ion channels has usually been demonstrated after biochemical purification of these proteins from native tissues. Maxi-K + channels are an example where the presence of a/3 subunit was not predictable. Given that maxi-K + channels found in 16 H.-G. Knaus, K. Folander, M. Garcia-Calvo, M. L. Garcia, G. J. Kaczorowski, M. Smith, and R. Swanson, J. Biol. Chem. 269, 17274 (1994). 17 H.-G. Knaus, M. Garcia-Calvo, G. J. Kaczorowski, and M. L. Garcia, J. Biol. Chem. 269, 3921 (1994).

[1 51

RECONSTITUT|ON OF CHANNELS INTO PLANAR BILAYERS

287

different tissues display distinct sensitivities to Ca 2+, this could be due to the presence of tissue-specific splice variants of the protein. However, it is now clear that coexpression of the/3 subunit with the pore-forming component markedly alters the Ca 2+ sensitivity of this channel. 1s'19 As well, the /3 subunit has profound effects on some of the pharmacologic properties of maxi-K + channels. Thus, the maxi-K +/3 subunit is an integral component of channel function, and by itself, will alter the Ca 2÷ sensitivity of the channel. Similar effects of/3 subunits have been found with other types of ion channels (e.g., voltage-gated Na, Ca 2+, and K + channels), and this phenomenon will undoubtedly be rediscovered as other ion channel families are explored. Biochemical purification of the maxi-K ÷ channel has helped us gain a better understanding of this protein's structure and function. Clearly, some of the ideas and techniques described in this article are directly applicable to other types of ion channel proteins, too.

~ M. Wallner, P. Meera, M. Ottolia, G. J. Kaczorowski, R. Latorre, M. L. Garcia, E. Stefani. and L. Toro, Recepr Channels 3, 185 (1995). 1~ O. B. McManus, L. M. H. Helms, L. Pallanck, B. Ganetzky, R. Swanson, and R. J. Leonard, Neuron 14, 1 (1995).

[15] R e c o n s t i t u t i o n o f N a t i v e a n d C l o n e d C h a n n e l s into Planar Bilayers By ISABELLEFAVRE,YE-MING SUN, and EDWARD MOCZYDLOWSKI Introduction a n d General Overview A planar bilayer is an artificial membrane formed across a small hole, - 5 0 / x m or larger in diameter. The hole on which the membrane is formed is usually placed in a thin plastic partition separating two aqueous compartments, but artificial bilayers may also be formed on a glass micropipette tip. Insertion or incorporation of a channel-forming molecule into such a membrane provides a simple experimental system for electrical recording of channel-mediated currents. Planar bilayer recording of ion channels is practiced for a number of reasons. Frankly, it is a technique that yields incredibly rich mechanistic information on a relatively low budget while offering kaleidoscopic displays of single-channel fluctuations that some workers find delightful, even soothing. More formally, the following applications and research stratagems have emerged as the principal rationales and justifications for forming planar

METHODS IN ENZYMOLOGY, VOL. 294

Copyright © 1999by AcademicPress All rightsof reproduction in any form reserved. 0076-6879/99 $30.00