J. Mol. Biol. (1992) 223, 929-948

Membrane Topology and Quaternary Structure of Cardiac Gap Junction Ion Channels Mark Yeager1v2t and Norton B. Gilula’ ‘Departments of Cell Biology and Molecular Biology The Scripps Research Institute, La Jolla, CA 92037, U.S.A. ‘Division of Cardiovascular Diseases Scripps Clinic and Research Foundation La Jolla, CA 92037, U.S.A. (Received 20 May

1991; accepted 24 September 1991)

The membrane topology and quaternary structure of rat cardiac gap junction ion channels antibodies containing czl connexin (i.e. (2x43) have been examined using anti-peptide directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved twodimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold elect,ron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic “loop” domain of at least M, = 5109 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native M, = 43,000 protein can be protease-cleaved on the cytoplasmic side of fragment. Western the membrane, resulting in an M, M 30,000 membrane-bound immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-27 1. Immunoelectron microscopy peptide is released after protease demonstrated that the M, x 13,900 carboxy-terminal cleavage and does not remain attached to the M, % 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the a1 polypeptide. Therefore, the M, x 13,900 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexons indicates conservation in the molecular design of the gap junction channels formed by a or fl connexins. Keywords: cardiac gap junctions; ion channels; membrane proteins; intercellular communication; peptide antibodies

1. Introduction

(termed

connexins

Cx$)

have

been

identified:

Gap junctions are specialized regions of contact between cells that enable them to exchange nutrient and signal molecules and thereby co-ordinate their metabolic and electrical activities (Loewenstein, 1981). Several different gap junction proteins

$ Abbreviations used: Cx, connexins; b1 = Cx32, M, = 32,000 rat liver gap junction protein; fi2 = Cx26, M, =

26,000 mouse liver gap junction protein; c(, = Cx43, M, = 43,000 rat car&&gap junction protein; PMSF, phenylmethylsulfonyl fluoride; KLH, keyhole limpet hemocyanin; PBS, phosphate buffered saline; TBS, Tris-buffered saline; BSA, bovine serum albumin: PTA, phosphotungstic acid

t Author to whom all correspondence should be addressed at: The Scripps Research Institute (MB6), 10666 North Torrey Pines Road, La Jolla, CA 92037,

1J.S.A. 929 0022-2836/92/040929-20

$03.00/O

a

h, = 32,000 protein in rat liver (Paul, 1986; Kumar & Gilula, 1986), a M, = 26,000 protein in mouse

0

1992 Academic

Press Limited

930

M.

Yeager and N. R. Cilula Liver (8,)

Heart (-,I

Extracellular gap Figure 1. Folding models for the M, = 43,900 heart (or,; also termed connexin 43) and Mr = 32,006liver (/III; also termed connexin 32) gap junction proteins. Amino acid sequences of the rat heart (Beyer et al., 1987) and liver (Paul, 1986; Kumar & Gilula, 1986) gap junction proteins were deduced from cDNA analysis. Boxed residues are identical in the 2 sequences. Hydropathy analysis predicts 4 membrane spanning domains. The predicted locations of the extracellular, membrane and cytoplasmic regions are indicated. Site-specific antibodies were generated to peptides L, (residues 101 to 112) and L, (residues 131 to 142) in the cytoplasmic “loop” region, C1 (residues 237 to 248) and Cz in the carboxy-terminal domain (residues 370 to 382) and E r (residues 51 to 65) and E, (residues 184 to 198) in the extracellular loops of 01~connexin. The arrow indicates the approximate site of cleavage by an endogenous protease in rat heart that generates a M, = 30,009 membrane-bound fragment. Amino-terminal antibodies generated to residues 1 to 20 and 6 to 17 have been characterized by Yancey et al. (1989) and DuPont et al. (1988), respectively. Beyer et al. (1989) examined the labeling of antibodies generated to peptide sequences 119 to 142 and 252 to 271. Antibodies to residues 314 to 322 were examined by El Aoumari et al. (1990). Laird & Revel (1990) have generated antibodies to peptides analogous to L, (i.e. residues 100 to 122), L, (i.e. residues 237 to 259), E, (i.e. residues 46 to 76), E, (i.e. residues 186 to 206) and C2 (i.e. residues 360 to 382). liver (Zhang & Nicholson, 1989), a M, = 43,000 protein in heart (Beyer et al., 1987) and a Mr = 70,000 protein in lens (Kistler et al., 1988). These gap junction proteins are derived from a multigene family that have been classified into two types, M and /?, based on the characteristics of their amino acid sequences (Risek et al, 1990; Gimlich et al., 1990). The p class of gap junction proteins was originally described in mammalian liver, with relative molecular masses of 32,000 (8, = Cx32) and 26,000 (/J, = Cx26). The initial protein described from the a class of gap junction proteins is the M, = 43,000 (aI = Cx43) protein found in mam-

malian myocardium. Northern blot analysis (Beyer et al., 1987) has suggested that the 01~ mRNA is expressed in rat heart, uterus, ovary and kidney. The structure of liver gap junctions containing PI and pz connexins has been thoroughly examined using electron image analysis (Unwin & Zampighi, 1980; Unwin & Ennis, 1984; Baker et al., 1985)) X-ray scattering (Caspar et al., 1977; Makowski et al., 1977), protease digestion (Zimmer et al., 1987; 1988) and immunochemical Hertzberg et al., labeling techniques (Milks et ol., 1988; Goodenough et aE., 1988). A model has emerged in which hexamerit oligomers (termed connexons) are formed by

Cardiac

Gap Junction

proteins that span the membrane four times such that the amino- and carboxy termini are located on the cytoplasmic side of the membrane (Milks et al., 1988; Hertzberg et al., 1988). Manjunath et al. (1985, 1987) demonstrated that cc1 connexin (M, = 43,000) is substantially larger than /?I connexin (A?, = 32,000). The availability of the amino acid sequences for the rat liver (Paul, 1986; Kumar & Gilula, 1986) and rat heart (Beyer et al., 1987) connexins allows a direct comparison of the gap junction proteins from these two tissues. Hydropathy analysis of the amino acid sequences predicts that both proteins contain four hydrophobic domains of sufficient length to span the lipid bilayer (Paul, 1986; Kumar & Gilula, 1986; Beyer et al., 1987; Milks et al., 1988). The a, and /I1 gap junction proteins contain the greatest sequence homology in the putative membrane spanning and extracellular domains. The /I1 gap junction protein has been extensively probed using site-specific peptide antibodies (Milks et al., 1988; Goodenough et al., 1988), and the immunolabeling results are consistent with the folding model shown in Figure 1. Here, we report an integrated biochemical and structural analysis of the tlr cardiac gap junction protein?. The membrane topology has been examined using peptide antibodies directed to seven different sites in the protein and cleavage by an endogenous protease in heart tissue. Our results confirm and extend previous topological analyses of t’he a1 protein using immunolabeling techniques and protease cleavage that support the folding model shown in Figure 1 (also see Table 1). In addition, we

931

Structure

isolation buffer contained solid phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co., St. Louis, MO) at a concentration of 1.0 mM. In I.0 mM-bicarbonate the pH was often less than 7, presumably due to hydrolysis of PMSF. Therefore, the pH of the bicarbonate buffer for the

(a)

4 C

5 6 N C

7 8 N C

9 N

10 11 12 13 C

N

provide the first analysis of the quaternary structure of a1 cardiac gap junctions using electron

microscopy and image processing. The density maps not only demonstrate that a1 connexons are hexamerit, but show that protease cleavage does not alter the hexameric subunit arrangement. 2. Materials

and Methods

(a) Isolation of cardiac gap junctions Rat ventricular gap junctions were isolated as described by Manjunath & Page (1986) (Fig. 2) from retired female breeder rats (Sprague-Dawley). This protocol is similar to others (Kensler & Goodenough, 1980; Gros et aZ., 1983) but involves fewer centrifugations and combines sarkosyl extraction with sucrose gradient centrifugation. The following modifications were made in the procedure. Two hearts were homogenized in 40 ml of isolation buffer for 1 min using a Tekmar tissuemizer (Cincinnati, OH) at full power. Buffer volumes were increased proportionately to allow isolation of junctions from 20 instead of 4 hearts. The wet weight was * 1 g/heart with a yield of -400 pg of gap junctions measured by a micro-protein assay (Pierce Biotec, Rockford, IL). Cardiac gap junctions are sensitive to proteolysis (Manjunath et al., 1985). Therefore, the 7 A preliminary the 39th Scientific Cardiology: Young (Yeager & Gilula,

report of this work was presented at Sessions of the American College of Investigators’ Awards Competition 1990).

Figure 2. SDS/gel electrophoresis profiles (a) and Western immunoblots ((b) and (c)) of rat ventricular subcellular fractions in the isolation of native (N) (lane 11) and cleaved (C) (lane 10) tlr gap junctions. When the protease inhibitor PMSF was present in the isolation buffers, the native CQ protein (N) with M, = 43,000 was isolated. In the absence of PMSF, an endogenous protease in heart tissue cleaved the a1 protein to a relative molecular mass of M, = 30,000. Lanes 1 and 13, protein molecular mass standards (Sigma Chemical Co., St. Louis, MO) (M, values are listed vertically adjacent to each band); lanes 2 and 3, crude membrane fractions after overnight extraction with potassium iodide to remove contractile proteins; lanes 4 and 5, supernatants after overnight potassium iodide extraction containing primarily contractile proteins; lanes 6 and 7, O/35 ye (w/v) sucrose gradient interface containing mainly mitochondria, sarcoplasmic reticulum and plasma membrane elements; lanes 8 and 9, sucrose gradient pellet with residual contractile and extracellular matrix proteins; lanes 10 and 11, 35/49 O/e sucrose gradient interface enriched for cardiac gap junctions; lane 12, isolated liver gap junctions (L). (b) and (c) are Western immunoblots corresponding to the gel lanes in (a). (b) Immunoblots probed with affinity-purified L, peptide antibodies (1: 100 dilution) showed binding to the native (M, = 43,000) and cleaved (M, = 30,000) polypeptides. Liver gap junctions (lane 12) were not labeled with these antibodies. (c) Immunoblots probed with preimmune immunoglobulins have no detectable binding.

M.

932

isolation of native a1 junctions was maintained using 50 mM-bicarbonate buffer.

Yeager and N. B. Gilula at -8

(b) Protease cleavage Cleaved tl, gap junctionsf were isolated by following the same protocol as for intact junctions, except that the isolation buffer contained 1.0 mM-bicarbonate and lacked PMSF (Manjunath et al., 1985). (c) Isolation

of liver gap junctions

Rat /31 gap junctions were isolated according to the alkali extraction protocol described by Hertzberg (1984) as modified by Zimmer et al. (1987) or the sarkosyl extraction protocol described by Manjunath & Page (1986). (d) SDSjpolyacrylamide

gel electrophoresis

Samples were solubilized at room temperature for -20 min and analyzed using a discontinuous buffer system (Hoefer Scientific Instruments, San Francisco, CA) with 4.5 y. (w/v) stacking gels and 11.5 y. separating gels as described by Laemmli (1970) using Protogel bisacrylamide (National Diagnostics, Manville, NJ). The following low relative molecular mass standards were used (Sigma Chemical Co., St. Louis, MO): bovine serum albumin, M, = 66,000; ovalbumin, M, = 45,000; glycerdehydrogenase, M, = 36,000; aldehyde-3-phosphate carbonic anhydrase, M, = 29,000; trypsinogen, M, = 24,000; soybean trypsin inhibitor, M, = 20,100; and lysozyme, M, = 14,200. With this system, the relative molecular mass of the native a1 protein was M, = 45,000, and the relative molecular mass of the protease-cleaved c(~ protein was M, = -30,000. (e) Generation

of afinity-puri$ed peptide antibodies

site-specijc

Generation of antibodies and affinity purification were as described by Milks et al. (1988). The following 6 oligo1985) with were synthesized (Houghten, peptides numbers indicating their position in the amino acid sequence of the CQprotein: L,, amino acid residues 101 to 112 (Arg Lys Glu Glu Lys Leu Asn Lys Lys Glu Glu Glu); L,, amino acid residues 131 to 142 (Glu He Lys Lys Phe Lys Tyr Gly Ile Glu Glu His); C,, amino acid residues 237 to 248 (Lys Asp Arg Val Lys Gly Arg Ser Asp Pro Tyr His); C,, amino acid residues 370 to 382 (Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile): E,, amino acid residues 51 to 65 (Ala, Phe, Arg, Cys, Asn, Thr, Gin, Gin, Pro, Gly, Cys, Glu, Agn, Val, Cys); and E,, amino acid residues 184 to 198 (Val, Tyr, Thr, Cys, Lys, Arg, Asp. Pro, Cys, Pro, His, Gln, Val, Asp, Cys). Peptides were conjugated to keyhole limpet hemocyanin (KLH) (Sigma Chemical Co., St. Louis, MO) using m-maleimidobenzoylN-hydroxysuccinimide ester (Liu et al., 1979; Green et al., 1982). Rabbits were immunized by injecting -200 pg of peptide-KLH in incomplete Freund’s Adjuvant (Sigma Chemical Co., St. Louis, MO) (1 : 1) subcutaneously on day 0 and 14 and finally -200 pg of peptide-KLH in 4 mg of alum (Sigma Chemical Co., St. Louis, MO) on day 21, t In this study, “cleaved” gap junctions contain connexin molecules that have undergone protease cleavage, as opposed t,o gap junction membrane bilayers that have been separated or “split” in the extracellular region.

intraperitoneally. Phlebotomy was performed 2 to 3 weeks after each injection. Affinity purification of the peptide antibodies was performed using the Acti-Disk system (FMC Bioproducts, Rockland, ME). Peptide (5 mg) dissolved in phosphate buffered saline (PBS = 10 m&%-phosphate; 150 mM-Nacl (pH 7.4)) were cycled through the Acti-Disk for - 1 h. The peptide solution was eluted, and unoccupied sites were blocked by cycling 10 ml of 100 mM-glycine (pH 7.4) through the disk. The disks were active after storage for several months at 4°C. A disk coupled with KLH was prepared in the same fashion. Serum (15 ml) was cycled through the Acti-Disk, and antibodies were eluted with 45 ml of 0.15 M-NaCl t,o which 180 ~1 of diethylamine (Sigma Chemical Co., St. Louis, MO) had been added. Any residual KLH antibodies were removed by passing the eluant through an Acti-Disk wit’h bound KLH. The peptide antibodies were dialyzed overnight against 7.5 mM-KCl. 0.5 mM-Tris (pH 7.5) at 4°C. concentrated lo-fold and then adjusted to a final concentrat,ion of I.5 mg/ml. Affinity-purified antibodies were used in all experiments. (f) lmmunoblot

andysis

Reactivity between the synthetic oligopeptides and the serum and affinity-purified antibodies was performed by applying l-p1 samples of peptide solutions in PBS to nitrocellulose strips (Schleicher and Schuell, Keene. NH). Reactivity was measured using an alkaline phosphatase immunosorbent assay (ProMega Biotec, Madison. WI). Immunoblotting was performed by transferring protein fractions from polyacrylamide gels onto nitrocellulosr (Schleicher and Schuell, Keene. XH) using an electrotransfer apparatus (Bio-Rad Chemicals, Richmond, CA) for 2 h at 55 mV (Towbin et al., 1979). The nitrocellulose blots were blocked with 3%) (w/v) bovine serum albumin (BSA; Boehringer-Mannheim Biochemicals, Indianapolis. IN) in Tris-buffered saline (TBS = 50 m&i-Tris, 300 mMNaCl (pH 7.5)) overnight at 4°C. The following steps were then performed sequentially: approx. 3 h incubation at room temperature with antibodies; 3 TBS washes for at least 1 h; incubation with lzsI protein A at dilution of 1 : 2000 (Amersham Corp, Arlington Heights, IL) fol m I h at room temperature or overnight at 4°C; 3 TBR washes for at least, 1 h. Autoradiography was then performed using Kodak XAR-5 film at -70°C with an intensifying screen. (R) Immunojluorescence

microscop?/

Cryofixation was performed by rapidly plunging freshly dissected rat heart or liver tissue into liquid nitrogen. A cryostat (International Equipment Corp.) was used to cut 4 to 5 pm thick sections. The sections were air dried on microscope slides, washed for 5 min in PBS and exposed to affinity-purified antibody solutions (diluted 1:25 to 1:lOO) for - 1 h at room temperature or overnight at 4°C. For competition experiments, 1.5 ~1 of peptide solution at a concentration of 1.0 mg/ml in PBS was mixed with 75 ~1 of antibody sample and then applied to the tissue. The slides were then washed in PBS for 20 min and incubated with fluorescein-conjugated goat anti-rabbit immunoglobulins (Cappel, West Chester. PA). Slides were again washed with PBS for 20 min. and sealed with a medium that retarded fading of the fluorescence (Johnson & Nogueira Araujo, 1981). Visualization of intercalated disks in phase contrast images was optimized by not fixing the tissue with ethanol

Cardiac

and

by

cutting

thinner

sections.

examined using a Zeiss Axiophot (h) Thin-section

Gap Junction

Tissue sections were microscope.

immunoelectron microscopy

All steps were performed at room temperature, unless otherwise indicated, using 100 ~1 samples containing -50 pg of x1 or /Ii gap junctions. Antibody penetration was facilitated by sonicating the membrane suspensions for 30 s (Kontes Micro-Ultrasonic Cell Disruptor, Hayward, CA). Samples were blocked with 3% BSA in TBS and then labeled with primary antibodies (diluted 1 : 10) for -2 h. followed by 3 TBS washes. The gap junctions suspended in TBS were then incubated for - 3 h with anti-rabbit immunoglobulins labeled with 5 nm gold particles (Janssen Life Sciences Products, Olen, Belgium) at a dilution of the gold antibodies (1 : 10) that optimized the labeling and minimized non-specific binding. Care was taken to prevent foaming during the pellet suspensions and incubations. The following steps were then sequentially performed: washing 3 times in TBS, fixation of membrane pellets for 1 h with 25% (v/v) glutaraldehyde (Polysciences. Inc., Warrington, PA) in 61 M-sodiumcacodylate buffer (pH 7.4), rinsing with 91 M-sodiumcacodylate, staining for 1 h with 1 o/o (v/v) 0~0, (Electron Microscopy Sciences, Fort Washington, PA) in (rl Msodium-cacodylate buffer, rinsing 3 times with 0.1 M-sodium-cacodylate buffer, rinsing with 0.05 Msodium-cacodylate buffer, 30 min exposure to 95% (w/v) tannic acid (Mallincktrodt) in 905 M-sodium-cacodylate buffer, 10 min rinsing with 1% (w/v) Na,SO, (J. T. Baker Chemical Co., Phillipsburg, NJ) in 61 iw-sodiumcacodylate buffer, 3 rinses (10 min) with Verona1 acetate buffer (pH 4.5) staining with uranyl acetate in Verona1 acetate buffer for 1 h, 2 rinses with Verona1 acetate buffer, and then 3 rinses in distilled water. The samples were progressively dehydrated in 30, 50, 70, 95 and 100% ethanol and finally propylene oxide (Polysciences, Inc., Warrington, PA). Embedding was performed by incubating the samples overnight in a 3 : 1 mixture of Epon (LADD Research Industries, Inc., Burlington, VT) and propylene oxide, followed by a -6 h incubation in 100% (v/v) Epon and finally overnight heating at 6OO”C in fresh 1000/b Epon. A diamond knife was used to cut thin sections, which were post-stained with lead citrate and uranyl acetate and then examined using a Philips CM12 electron microscope.

crystallization gap junctions

(i) Two-dimensional

of cardiac

The gap junction-enriched 35/49% sucrose gradient fractions (Fig. 2, lanes 10 and 11) were retrieved and transferred to Spectra/Par (Spectrum Medical Industries, Inc., Manville, NJ) dialysis tubing. Crystals suitable for electron image analysis were obtained by sequential 12 to 24 h dialysis against 05% (w/v) sodium deoxycholate (Sigma Chemical Co., St. Louis, MO) and then 905% (w/v) dodecyl-fi-n-maltoside (Cal Biochem Corp., La Jolla. CA) in a buffer containing 5.0 miw-Hepes (Sigma Chemical Co., St. Louis, MO) (pH 8), 0.1 mM-CaCl, (J. T. Baker Chemical Co., Phillipsburg, NJ), 95 miw-MgCl, (J. T. Baker Chemical Co.. Phillipsburg, NJ) and 902% (w/v) sodium azide (Sigma Chemical Co., St. Louis, MO).

(j) Electron

microscopic

Samples of the crystalline with either 246 (w/v) uranyl

image

analysis

gap junctions were acetate (Ted Pella,

stained Tustin.

Structure

933

CA) or 2% (w/v) phosphotungstic acid (PTA) (Sigma Chemical Co., St. Louis, MO) (pH adjusted to 5 with potassium hydroxide). Images were recorded under minimum electron dose conditions (10 to 20 electrons/A*: Williams & Fisher, 1970) using a Philips EM4OOT. Bn optical diffractometer was used to select images for data processing that showed the sharpest reflections with uniform staining, minimal astigmatism and minimal drift. Evaluation of the Fourier transform indicated that the degree of defocus was 5500 to 6500 A (1 A = 6 1 nm) so that the contrast transfer function was uniform and of a constant sign to 17 A resolution (Erickson & Klug, 1971). Micrographs were digitized with a microdensitometer (Perkin-Elmer Corp., Eden Prairie, MN) using step and aperture sizes of 25 pm. The extraction and refinement of the crystal lattice parameters and sinc-weighted amplitudes was as described (Amos et al., 1982). Lattice distortions were corrected using the procedures described by Henderson et al. (1986). Two-dimensional projection maps were calculated by Fourier transformation using the amplitudes and phases corrected for lattice distortions (Yeager, 1987, 1989).

3. Results (a) Isolation of cardiac gap junctions containing native and cleaved polypeptides The u1 protein is progressively enriched during the isolation procedure (Fig. 2(a)). When cardiac gap junctions were isolated in the presence of the protease inhibitor PMSF, the native protein migrated with an apparent M, of N 45,000 (lane 11 in Fig. 2(a)), close to the value of 43,000 deduced from a cDNA analysis (Beyer et al., 1987). When PMSF was excluded from the isolation buffers, an endogenous protease in heart tissue cleaved the polypeptide to M, = 29,000 to 31,000 (lane 10 in Fig. 2(a)). Isolated gap junction plaques containing either native or cleaved protein were used for the structural analysis using immunochemistry, protease cleavage and electron crystallography. (b) Peptide

antibodies speci$cally bind to a, peptides and c(~ connexin

The folding model in Figure 1 displays the predicted locations of the peptide sequences to which antibodies were generated. Sites L,. I,, and C, reside close to the membrane surface, whereas site Cz is located at the extreme carboxy terminus. Sites E, and E, reside in the extracellular loops. The dot immunoblots in Figure 3 show that the affinity-purified peptide antibodies specifically bound to their corresponding peptides. The only evidence for cross-reactivity was that some preparations of L, antibodies also bound to the L, peptide as demonstrated in the over-exposed dot immunoblot of the L, antibodies in Figure 3. To test for potential cross-reactivity between the peptide antibodies and other antigens in heart tissue, Western immunoblots were prepared for each of the peptide antibodies and the subcellular fractions obtained from the isolation of cardiac gap junctions. The immunoblot in Figure 2(b) shows that peptide anti-

934

M. Antibodies

Peptide h

E,

Immune Preimmune

~~

Immune Prelmmune

Yeager and N. B. Gilula

L2

-

‘?

__(_

C2

_d.”

El

E2

.~

Figure 3. Immunodot blots of affinity-purified peptide antibodies (1 : 50 dilution) prepared against peptides L,, L,. C, and C, in the cytoplasmic domains and E, and E, in the extracellular domains of the a1 protein (see Fig. 1). The antibodies bound specifically to their respective synthetic peptide. Some preparations of L, antibodies labeled both the L, and L, peptides. Preimmune immunoglobulins (1 : 50 dilution) did not bind to the peptides. body L, specifically labeled t’he cleaved and native c1i proteins in the partially enriched membrane fractions (lanes 6 and 7, respectively) and the fractions highly enriched in gap junctions (lanes 10 and 11). Faint labeling of higher relative molecular mass polypeptides is most readily explained on the basis of multimeric forms of the a1 native (M, = 43,000) and cleaved (M, = 30,000) polypeptides. Strong labeling of a monomeric M, = 70,000 polypeptide, as observed by Harfst et al. (1990), was not evident in our preparations. No significant binding was detected to other proteins in the cell fractions. Labeling of the c1r protein was not detected in the crude membrane fractions (lanes 2 and 3) presumably because the amount of gap junction protein in those fractions was too low to be detected by the “‘1 protein A label. No binding was detected to liver gap junction protein (lane 12), and none of the proteins was labeled by preimmune immunoglobulins (Fig. 2(c)). Immunoblots similar to Figure 2(b) and (c) were prepared for peptide antibodies L,, C,, C,, E, and E, (data not shown), and these peptide antibodies also bound specifically to the ai protein. (c) Immunohistochemical localization antibodies

of peptide

Immunohistochemical localization of the sitespecific peptide antibodies was examined using phase contrast and indirect immunofluorescence microscopy (Fig. 4). Gap junctions in the heart are known to reside in the intercalated disks (McNutt & Weinstein, 1970). In longitudinal cryo-sections of rat heart muscle cells, the intercalated disks appear as discrete high contrast lines readily distinguished from the thin striations of the muscle fibrils (see top portion of Fig. 4(a)). The corresponding indirect immunofluorescence micrograph (Fig. 4(b)) displays fluorescent labeling in discrete linear patterns corresponding to the location of intercalated disks. When the muscle cells are sectioned obliquely (see bottom portion of Fig. 4(a) and (b)), the intercalated disks

are not readily identified in the phase image. The corresponding area in the fluorescence images displays a “speckled” pattern. Phase contrast and immunofluorescence micrographs for the cytoplasmic peptide antibodies are shown in Figure 5. For antibodies L,, L, and C, the cryo-sections are primarily along the longitudinal axis of the cells, and the linear fluorescent signal corresponded to the location of the intercalated disks in the phase images. For the C, antibodies, the cryo-section was more oblique and resulted in a more “speckled” pattern of fluorescence. Our initial expectation was that the extracellular domains would not be exposed during cryosectioning. We were surprised that’ the extracellular antibodies did in fact specifically label intercalated disks (Fig. 6). At low magnification, the fluorescence was not uniformly distributed over the entire tissue section but tended to have a patchy distribution with a higher overall diffuse background compared with the cytoplasmic antibodies (Fig. 5). Competition experiments were used to verify that the patchy labeling was specific for intercalated disks. In the absence of peptide (denoted (-) in Fig. 6), the linear fluorescent signal for the E, and E, antibodies corresponds to t’he intercalated disks in the phase images. When the E, and E, peptides were added to their respective antibody solutions (denoted (+) in Fig. 6), the linear fluorescent signal was largely extinguished. In summary, the affinity-purified antibodies generated to peptides predicted to reside in the cytoplasmic and extracellular domains of the ai protein bound specifically to the regions known to contain gap junctions. Immunofluorescence microscopy using preimmune immunoglobulins showed mild autofluorescence and no specific labeling (data not, shown). (d) Ultrastructural

localization

of peptide

antibodies

Further evidence for specificity of the peptide antibodies was obtained by immunogold labeling of isolated cardiac gap junctions. Thin-section electron microscopy revealed that gap junctions, identified by their double membrane profiles (Fig. 7), were heavily decorated on their cytoplasmic surfaces with colloidal gold particles bound to L, antibodies. Specificity of antibody labeling was indicated by minimal labeling of contaminating amorphous material and non-junctional single membrane structures (small arrows in Fig. 7). Interestingly, the micrograph displays occasional splitting of the extracellular gap regions at the ends of the membrane sheets (large arrows in Fig. 7). Such splitting may be due to weaker bonds linking the polypeptides across the extracellular gap compared with the strength of the hydrophobic interactions within the bilayers. This is consistent with the harsh, denaturing conditions, which split liver gap junctions but do not appear to affect the lipid bilayer structure (Manjunath et al., 19846; Milks et al., 1988; Goodenough et al., 1988).

Cardiac Gap Junction Structure

Fig antibs sectia readil interc of tht variec brigh

935

;ure 4. Phase contrast micrograph (a) and indirect immunofluorescence micrograph (b) for C, site-specific pep Itide odies (1 : 100 dilution) directed against the carboxy-terminal peptide in the GINprotein (see Fig. 1). In longitud linal Ins (top portion of (a)), the high contrast linear striations (large arrows) correspond to the intercalated disks that ; are ly distinguished from the striations of the sarcomeres (small arrows). In oblique sections (bottom portion of (a)) the :alated disks are not readily identified. The correlated fluorescence (b) and phase contrast (a) images show binc ding ? antibodies to the intercalated disks in which the gap junctions are known to reside. The fluorescence inten sity 1 between different preparations of antibodies generated against the same peptide. For instance, the labelir Lg is ter in this preparation of C, antibodies compared with the preparation used in Fig. 5. Scale bar represents 25 Pm.

M.

Yeqer

and N. B. Gil&a

Figure 5. Phase contrast micrographs (a) and indirect immunofluoreseence micrographs (b) for site-specific peptide antibodies L,, L,, C, and C2 directed to cytoplasmic domains in the CI~protein (see Fig. 1). The correlated fluorescence and phase contrast images show binding of the antibodies to the intercalated disks in which the gap junctions are known to reside. The antibodies were diluted 1 : 25 except for the L, antibodies which were diluted 1 : 10. Scale bar represents 200 pm.

Cardiac Gap Junction Structure

937

Figure 6. Phase contrast micrographs (a) and indirect immunofluorescence micrographs (b) for site-specific peptide antibodies E, and E, (1 : 25 dilution) directed to the extracellular domains in the at protein (see Fig. 1). In the absence of peptide added to the antibody solution (denoted( -)), the fluorescence and phase contrast images show binding of the antibodies to the intercalated disks in which the gap junctions are known to reside. When the E, and E, peptides are added to their respective antibody solutions (denoted (+)), the fluorescent signal is largely extinguished. Scale bar represents 100 pm.

938

M.

Yeager and N. B. Gilula

I 3gure 7. Immunogold labeling of cardiac gap junctions by peybtide L, in the tlI protein (see Fig. 1). The absence of gold

site-specific peptide antibodies (1 : 10 dilution) ag:Cnst label on single bilayer membranes (small arrows) and am orphous material (periphery of the image) indicates the specificity of the antibodies for cardiac gap jund Sons conkaining tll connexin. Interestingly gap junction sheets display occasional splitting of the extracellular gap regic m at the edges of the sheet (large arrows). Scale bar represents @l pm.

Gap junctions containing native and proteasecleaved polypeptides were labeled by antibodies L, , L, and C,; however, only native junctions were labeled by antibody Cz (Fig. 8). The minimal labeling of cleaved junctions incubated with Cz antibodies was comparable to the appearance of junctions incubated with preimmune immunoglobulins (data not shown). In addition, tlr gap junctions were not significantly labeled by the extracellular antibodies E, and E, (data not shown).

(e) Peptide

antibody

binding to native and cleaved ml connexin

The immunoblots in Figure 9 show the binding of the four cytoplasmic u1 site-specific peptide antibodies to native cur protein (M, = 43,000 with degradation products at M, = 35,000 and 33,000), cleaved a, protein (M, = 30,000) and the /I1 protein (M, = 32,000). Corresponding immunoblots for the extracellular a1 antibodies are shown in Figure 10. The interpretation of Figures 9 and 10 is facilitated by referring to the topological models of a1 and p1

connexins in Figure 1. The immunoblots in Figures 9(a) and 10 show that peptide antibodies L,, L,, C,, E, and E, bind to the cleaved tli protein. In contrast, peptide antibody Cr only bound to the native a1 protein. Therefore, the site of cleavage must be located between peptides C, and Cr. The the approximate arrow in Figure 1 identifies cleavage site that would generate a M, = 30,000 membrane-bound fragment. If the cleavage site is indeed located in this region, then antibodies generated to residues 252 to 271 should bind to both native and cleaved a1 connexin. The immunoblot in Figure 11 confirms this expectation. As expected, peptide antibodies L,, L,, C,, E, and E, bind to the native u1 protein. Although the C, peptide antibodies did label both native and protease-cleaved a1 protein, in several experiments there appeared to be preferential binding to the cleaved a1 protein as shown in Figure 9. It is noteworthy that none of the peptide antibodies showed significant cross-reactivity with the /I1 liver protein (Figs 9 to 11, lanes labeled L). Control immunoblots immunoglobulins showed no using preimmune specific protein binding.

Cardiac

939

Gap Junction Structure

Native

(a) Figure 8. Immunogold labeling of native (a) and antibodies L,, L,, C, and C, (1 : 10 dilution) directed protease-cleaved gap junctions are decorated by antibodies. Thus, the carboxy-tail epitope for the C, Scale bar represents @2 pm.

Cleaved

(b) protease-cleaved (b) cardiac gap junctions by site-specific peptide to cytoplasmic domains in the ai protein (see Fig. 1). All native and the antibodies except for cleaved junctions incubated with C, antibodies is released after protease cleavage (see model in Fig. 1).

M.

940

C

N

L

C

N

L

C

N

L

C

Yeager and N. R. G&la

N

252 to 271 C N L

L

43,000 20,000

(b) 43,000 30,000

Figure 9. Immunoblots

of native (N) and proteasecleaved (C) cardiac and liver (L) gap junction proteins using site-specific peptide antibodiek directed against cytoplasmic sequences in the a1 cardiac gap junction protein. The immunoblots in (a) were exposed to sitespecific peptide antibodies L,, L,, C, and C,, respectively diluted 1: 100, 1: 100, 1 : 50 and 1 : 25. The 4 peptide antibodies do not bind to liver gap junctions (L). The immunoblots in (b) were exposed to preimmune immunoglobulins and show no labeling of the gap junction proteins. Each lane contains 20 pg protein.

(f) Variability in antibody labeling immunojluorescence, immunoblotting immunoelectron microscopy

by and

Comparison of the signal intensities in Figures 4 to 11 indicates variability in the binding of the peptide antibodies to the a, protein. For example, by indirect immunofluorescence (Fig. 5) the relative

Pr&mmma C

N

L

C

N

IllWlWM

L

Pnimmunr

CNLCNL

Mr

Figure 10. Immunoblots of native (N) and proteasecleaved (C) cardiac and liver (L) gap junction proteins using site-specific peptide antibodies directed against extracellular sequences. E, and E,, in the a1 cardiac gap jun&ion protein. The peptide antibodies (1 : 30 dilution) label both native M, = 43,000 and protease-cleaved M, = 30,000 cardiac gap junction protein but do not bind to liver gap junction protein (L). Preimmune immunoglobulins do not bind to any of the gap junction proteins. Each lane contains 20 pg protein.

Figure 11. Immunoblot of native (N) and proteasecleaved (C) cardiac and liver (L) gap junction proteins using antisera containing antibodies directed against’ amino acid residues 252 to 271 in the 01~ cardiac gap junction protein. The antiserum was a generous gift of Dr Eric Beyer. The antibodies bind to native M, = 43,000 and proteolytic fragments of the cardiac gap junction protein, but do not bind to liver gap junctions (L). Each lane contains 20 pg protein.

signal strength listed in decreasing intensity was as follows: L, > C, > L, > C&. However, variability of signal intensity was observed in different preparations of the same antibodies. For example, different antibody preparations were used for the G immunofluorescence micrographs shown in Figures 4 and 5, and the signal is substantially brighter in Figure 4. Immunoblots (Fig. 9) also displayed variability in the binding of the cytoplasmic peptide antibodies to the a, protein as follows: L, - C, > L, > C,. However, for this immunoblot the Cz antibodies were diluted 1 : 25 versus 1 : 100 for the L, and L, antibodies. As noted above, some preparations of C1 antibodies also showed stronger labeling of the native M, = 43,000 protein than displayed in Figure 9. The extracellular E, antibodies displayed a strong signal by both immunofluorescence (Fig. 6) and immunoblotting (Fig. 10). Although specific labeling of int,ercalated disks and gap junction protein was observed with the Ez antibodies, the signal was substantially weaker than for the E, antibodies, and the non-specific background was higher. For immunoelectron microscopy (Fig. 8), adequate decoration by colloidal gold particles was achieved only by using peptide antibody concentrations five to tenfold higher than for immunoblotting or immunofluorescence. Nevertheless, the immunogold labeling was specific for gap junctions. The variability in the binding of the peptide antibodies to immunodetected by their epitopes as histochemistry, immunoelectron microscopy and immunoblotting presumably relates to conformational differences in the presentation of the epitope

Cardiac Gap Junction Structure

Cleaved

Notiw

Figure 12. Images of native and protease-cleaved either uranyl acetate or phosphotungstic acid (PTA). to deoxycholate and dodecyl-fi-n-dodecylmaltoside. optical diffraction. Scale bar represents 0.1 pm

941

2-dimensional crystalline arrays of GINgap junctions stained with The hexagonal packing of the oligomers was induced by exposure Asterisks (*) define the boxed areas judged most. crystalline by

to the antibodies under these different conditions as well as variable accessibility of the epitopes in tissue sections, isolated junctions and junction protein on nitrocellulose paper.

(g) Electron microscopic image analysis Electron microscopic image analysis of native and protease-cleaved cardiac gap junctions was carried out to image directly the molecular structure of the channel. Figure 12 displays two-dimensional crystalline arrays of native and protease-cleaved a1 gap junctions stained with either uranyl acetate or PTA. Cardiac gap junction plaques typically contained only a few unit cells that exhibited hexagonal packing. Dialysis against deoxycholate and then dodecyl-/-I-n-dodecylmaltoside resulted in coherently diffracting domains that contained 300

to 600 unit cells. The calculated Fourier transforms in Figure 13 display hexagonal symmetry extending to -20 A resolution. It is apparent by viewing the images in Figure 12 edge-on that the crystalline domains have lattice disorder. By correcting for crystal lattice distortions according to the methods described by Henderson et aZ. (1986), the digitized transforms showed weak reflections to a resolution of 16 A with a phase error of < 18”. The principle effect of the correction for the crystal lattice distortions was to reinforce the sixfold modulations of the protein subunits in the Fourier syntheses without any imposed symmetry. Since the oligomers display hexagonal symmetry, the projection maps in Figure 14 were calculated using p6 plane group symmetry. Within the accuracy and resolution of the data, the projection maps of native and cleaved gap junction crystals are not significantly different.

942

M.

Yeager and N. B. G&la

Native

Cleaved

C 0

f

iif

Figure 13. Fourier transforms of digitized micrographs from crystalline arrays of a, gap junctions shown in Fig. 12. Although the computed diffraction patterns of the native and protease-cleaved junctions are comparable, the patterns observed in uranyl acetate and PTA stains are quite different due to differential staining (see Fig. 14).

4. Discussion We have used complementary approaches to examine the tertiary folding and quaternary structure of the rat cardiac (ar) gap junction protein: immunoblotting with seven site-specific peptide antibodies, cleavage by an endogenous protease in heart and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Potential pitfalls in using antibody labeling techniques for discerning membrane protein topology have been pointed out by McCrea et al. (1987). For example, conflicting results using immunolabeling techniques have been obtained for the topology of the acetylcholine receptor. To ensure that the antibodies we have used are reliable labeling reagents, we have rigorously verified antibody specificity using several, complementary immunolabeling

techniques: dot immunoblotting, Western immunoblotting, immunofluorescence microscopy and immunoelectron microscopy. The concordant results from these experiments using different immunolabeling methodologies provides compelling evidence favoring the model in Figure 1. In general, our results confirm and extend prior immunochemical and protease cleavage analyses of a1 connexin (Table 1) that have appeared during the course of our experiments. In our analysis, the synthetic peptides were substantially shorter (12 to 15 residues versus 22 to 30 residues) than those used by others. The binding “footprints” of our antibodies on the protein are therefore smaller and will allow finer mapping of functionally important domains in future physiological experiments. In addition, polyclonal antibodies were affinity-purified against their respective peptides to minimize non-specific binding. The M, = 43,000 a, poly-

Cardiac Gap Junction

Structure

Native

943

Cleaved

Figure 14. Two-dimensional projection maps of native and protease-cleaved aI gap junctions stained with uranyl acetate or phosphotungstic arid (PTA) The maps display protein subunits (continuous contours) arranged in hexagonal oligomers that form central channels. The hexagonal channel structure is not detectably altered by protease cleavage. Uranyl acetate penetrates the ion channel (circular broken contours), whereas PTA defines the outer contour of the hexamers. and is comparatively excluded from the channel. The center-to-center separation between the hexameric oligomers is 85 A. (Separation between fiducidal marks is 20 A).

peptide appears to be the major, if not the only, component of rat native cardiac gap junctions. A cytoplasmic “loop” domain of at least M, = 5100

(residues 101 to 142) and the cytoplasmic carboxyterminal domain are readily accessible for peptide antibody labeling. Protease cleavage of the native M,= 43,000 protein releases the M, = N 13,000 soluble carboxy-terminal peptide from a M, = - 30,000 membrane-bound fragment (Manjunath et al., 1987). Immunoblot analysis demonstrates that the site of protease cleavage resides between residues 252 and 271. Electron microscopic image analysis of two-dimensional membrane crystals shows that the ~1~polypeptides are arranged in hexameric oligomers, analogous to the quaternary arrangement of liver gap junction

connexons

containing

/II1 or /I2 connexins.

Release of

the carboxy-terminal AZ, = N 13,000 from tll connexin by protease cleavage does not detectably alter the hexameric subunit arrangement. (a) Membrane topology de&ed by peptide antibody labeling and protease cleavage Five peptide antibodies were directed to sites predicted to reside in the cytoplasmic domains of the a, protein (Fig. 1). The L, and L, peptides are close to the membrane surface in the cytoplasmic loop flanked by the second and third membrane spanning domains. Since peptide antibodies L, and L, both bind to the native and cleaved u1 protein, the distance between these sites allows one to esti-

M. Yeager and N. B. Gil&a

944

Table 1 of antibodies to the CC1 cardiac gap junction

Characterization

Antibody Peptide antigen (label) l-20

Binding to protein by Western blotting

Topology

characteristics

Affinity

purified

and binding results

Binding to intercalated disks by immunofluorescence

Binding to isolated junctions by immunoelectron microscopy

+++

+++

Amino terminus

Affinity

protein

+++

Reference Yancey et al. (19893

Serum

purified

5-17

Amino-tail

+++ Affinity purified

? ? ?

? 1 ?

DuPont et al. (1988)

4676 (EL-46)

Extracellular loop M, to M,

+++ Affinity purified

‘2 ‘! ?

? ? ?

Laird & Revel (1990)

51-65 @I)

Extracellular loop M, to M,

+++ Affinity purified

loo-122 (CL-100)

Cytoplasmic loop

+++ Affinity purified

101-l 12 (L)

Cytoplasmic loop

+++ Affinity purified

119-142

Cytoplasmic loop

131-142 (Ld

--Affinity purified

This study

+++ Affinity purified

Laird & Revel (1990)

+++ Affinity purified

+++ Affinity purified

This study

+++ Serum

+++ Serum

+++ Serum

Cytoplasmic loop

+++ Affinity purified

+++ Affinity purified

+++ Affinity purified

Gwt junctions

Cytoplasmic loop

+++ Affinity purified

+++ Affinity purified

+++ Serum

184-198 (E,)

Extracellular loop M, to M,

+++ Affinity purified

+++ Affinity purified

186206 (EL-186)

Extracellular loop M, to M,

+++ Affinity purified

237-248 ((J,l

Cytoplasmic carboxy-tail

+++ Affinity purified

237-259 (CT-237)

Cytoplasmic Carboxy-tail

+++ Affinity purified

! ? ‘f

? I ?

Land & Revel (1990)

252-27 1

Cytoplasmic carboxy-tail

+++ Serum

+++ Serum

+++ Serum

Beyer et al. (1989)

314-322

Cytoplasmic carboxy-tail

+++ Affinity purified

+++ Affinity purified

+++ Affinity purified

El Aoumari

36CG382 (CT-3601

Cytoplasmic C terminus

+++ Afinity purified

+++ Affinity purified

Laird & Revel (1990)

376382

Cytoplasmic C terminus

+++ Affinity purified

+++ Affinity purified

This study

Gl + + + = - - = ? ? 1= t Isolated

experiment experiment experiment rat cardiac

+++ Affinity purified ? 1 ?

2 ( !

+++ Affinity purified

performed with positive result. performed with negative result. not performed or not reported. gap junctions served as the antigen, rather than a synthetic

mate the size of the cytoplasmic loop. Yancey et al. (1989) have estimated a minimum size for the loop of M, = 4100 based on protease digestion experiments. Since peptide antibodies L, and L, span the range of amino acid residues 101 to 142, we estimate that the minimum size of the cytoplasmic loop is M, = 5100. Beyer et al. (1989) generated antibodies

This study

Yancey et al. (1989) This study

Affinity

purified

Affinity

purified

1 I ‘1

+++ Affinity purified

Beyer et al. (1989)

Laird & Revel (1990)

+++ Affinity purified

This study

et al. (1990)

peptide

to a peptide (residues 119 to 142) in the mid-portion of the cytoplasmic loop. That serum labeled the a, protein on immunoblots and decorated the cytoplasmic surfaces of isolated gap junctions in thin sections. A different polyclonal antibody prepared against isolated a, gap junctions also had a binding site in the loop region (Yancey et al., 1989). Thus,

Cardiac

Gap Junction

virtually the entire polypeptide in the cytoplasmic loop domain appears to be readily accessible, and the small amino-terminal and large carboxyterminal cytoplasmic domains do not shield this domain from the bulk aqueous environment. The model in Figure 1 predicts that peptide antibody C, will bind to a site close to the membrane surface. Interestingly, the C, peptide antibodies appeared to display preferential binding to the cleaved a1 protein in several immunoblots (Fig. 9). However, by immunoelectron microscopy the C, antibodies showed comparable labeling of gap junctions containing native and cleaved protein (Fig. 8). Presumably, a structural change in the carboxyterminal domain after exposure to SDS-containing buffers during immunoblotting interferes with or alters the binding of peptide antibodies to site C,. Site-specific peptide antibody CZ is directed against the extreme carboxy-terminal peptide. The immunoelectron micrographs in Figure 8 and the immunoblots in Figure 9 show that this antibody labeled only the native and not the cleaved protein. Thus, protease cleavage releases a M, = - 13,000 soluble carboxy-terminal fragment(s), and the site cleavage must be located somewhere between peptides C, and C,. (Since the solubilized cytoplasmic fragment was not recovered, there may be several cleavage products that sum to M, = - 13,000.) The arrow in the folding model of the a1 protein (Fig. 1) indicates the approximate location between C, and C, that would generate the M, = - 13,000 carboxy-terminal fragment(s) and the amino-terminal M, = 30,000 membrane-bound, fragment containing the binding sites for peptide antibodies L,, L,, C,, E, and E,. The site of cleavage has been further delineated using antibodies generated to residues 252 to 27 1. Since these antibodies label both the native and cleaved a, polypeptides (Fig. ll), the site of protease cleavage must reside between residues 252 to 27 1. The amino-terminal domains of the ai and pi proteins are highly homologous (Fig. 1) (Nicholson et al.. 1985), and Western immunoblots show that site-specific antibodies to the amino-terminal sequence of /11 connexin (Zervos et al., 1985) can also bind to a1 connexin. Yancey et al. (1989) have shown that the amino-terminal sequence of the ai protein is accessible on the cytoplasmic surface for immunogold labeling by site-specific antibodies. Thus, the amino-terminal domain, carboxyterminal domain and the loop domain are all accessible on the cytoplasmic side of the membrane. The extracellular domains have been probed using antibodies E, and E,. Isolated gap junctions could not be labeled by E, and E, using thin-section immunogold electron microscopy. This is consistent wit,h the known narrowness of the extracellular gap region that would preclude penetration of antibody probes. Interestingly, intercalated disks were labeled by E, and E, in cryo-sections of heart muscle. Over the surface of the cryo-section, a pat,chy distribution of fluorescence was observed, presumably because the surface area accessible for

945

Structure

labeling cellular plasmic

is at least a factor of 2 less for the extragap domains compared with the cytodomains.

(b) Cardiac

gap junction liver

antibodies

gap junction

do not bind to

protein

As shown in Figure 1, the proposed folding of a1 connexin resembles that of /I1 connexin (Beyer et al., 1987; Milks et al., 1988). The greatest amino acid sequence homology between these two proteins is in the extracellular loops and the transmembrane domains. Although the model predicts a similar topology for the folding of the cytoplasmic domains, none of the a1 cytoplasmic peptide antibodies crossreacts with the fil protein (Figs 9 to 1 1 ), even though peptides L,, L, and C, have some regions of identity to the /I1 protein (Fig. 1). sequence the cytoplasmic loop and carboxyTherefore, terminal domains of the a1 protein have unique immunoreactive properties that distinguish it from the fll protein. Based on the sequence homology between the a1 and fli extracellular domains (Fig. l), one would expect cross-reactivity between the extracellular E, and E, antibodies and the /Ii protein. However, immunoblots for the extracellular antibodies did not display cross-reactivity with the b1 protein (Fig. 10). Nevertheless, preliminary immunofluorescence experiments using extracellular antibodies generated to the /3i protein display binding to intercalated disks in cryo-sections of heart. In addition, immunoblots using antibodies generated to an extracellular peptide (residues 164 to 189) in the /?I protein displayed weak labeling of the a, protein (Beyer et al., 1989). (c) Hexameric

structure

of the cardiac

gap junction

ion channel

Electron microscopic image analysis demonstrates that the ion channel is formed by a hexamerit arrangement of a1 polypeptides. Previous work using membrane-impermeable enzymes as probes of protein topology has not demonstrated that protease cleavage does not alter the quaternary structure of the protein, which could potentially affect the pattern of peptides generated by enzymatic cleavage. The density maps in Figure 14 demonstrate that the structure of native cardiac gap junctions is not altered detectably by cleavage of the al protein from M, = 43,000 to 30,000 (Fig. 14). Therefore, the Mr = - 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The absence of immunogold labeling of cleaved a, junctions incubated with C2 antibodies (Fig. 8) demonstrates that the M, = - 13,000 carboxy-terminal peptide is released after cleavage and does not remain attached to the M, = 30,000 membrane bound fragment via non-covalent interactions. Interestingly, the images in Figure 12 of the native and cleaved a1 gap junctions stained with uranyl acetate demon-

M.

Yeager and N. B. Gilula

strate that the oligomers and the channel are seen more clearly after protease cleavage. The increased clarity of the connexons is probably due to the loss of the cytoplasmic domain with reinforcement of the channel image. The cytoplasmic polypeptide appears to obscure the channel structure in the images of the native crystals and presumably corresponds to the “fuzzy” layer on the cytoplasmic surface seen in thin-sectioned images (Manjunath et al., 1984a). Additional mass on the cytoplasmic surface of a1 gap junctions has also been demonstrated by the deep etching, freeze-fracture technique (Shibata et al., 1985). There are at least two possible explanations why the loss of the M, = w 13,000 carboxy-terminal peptide does not alter detectably the hexameric structure. The first is that the electron microscopic image analysis reinforces only those parts of the structure that are crystalline. If the carboxy-terminal domain is disordered, then it will not be detectable in the density maps. A second explanation is that if the carboxyterminal is positioned approximately in line with the transmembrane and extracellular domains, then the loss of this mass will not alter detectably the two-dimensional projection maps. (d) Implications

for the structure of gap junctions and membrane channels

The hexamerie structure of cardiac gap junction ion channels supports the concept that ion channels are formed by membrane protein oligomers (Unwin, 1989) as has been found for hexameric gap junctions formed by /3 connexins (Unwin & Zampighi, 1980; Unwin & Ennis, 1984; Baker et al., 1985) and the pentameric acetylcholine receptor (Toyoshima & Unwin, 1988; Mitra et al., 1989). The similar quaternary structure between gap junctions containing a and p connexins indicates conservation in the molecular design of gap junction channels. However, the lack of sequence homology and difference in size of the cytoplasmic loop and carboxyterminal domains of a1 and /I1 connexins, as well as the absence of cross-reactivity between the cardiac gap junction peptide antibodies and f3i protein, suggests considerable divergence in the cytoplasmic loop and carboxy-terminal domains of a and /3 connexins. Such diversity may confer unique functional properties for different connexin proteins. For example, the unique structure of the cytoplasmic domains of the cardiac gap junction protein may be related to a potential regulatory role in mediating cardiac conduction (Barr et al., 1965; De Mello, 1982) and arrhythmias (Spach, 1983). We thank Dr Richard Houghten for preparing the synthetic peptides and Dr Linda C. Milks for assistance with preparation of the peptide antibodies. In addition, we acknowledge the skillful technical assistance of John Williamson, Horatio Kido and Gina Lento. M.Y. thanks Dr Curtis Wilson for guidance in performing immunofluorescence microscopy. We also thank Dr Eric Beyer for providing serum containing antibodies to residues 252p

271. This research 37904 (awarded to Award HLO-2129 Center American #%31153 (awarded

was supported by NIH Grant GM N.B.G.), a NIH Clinical Investigator (awarded to M.Y.) and a National Heart Association Grant-in-Aid to M.Y.).

References Amos, L. A., Henderson, R. & Unwin, P. N. T. (1982). Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Progr. Biophys. Mol. Biol. 39, 183-231. Baker, T. S., Sosinsky, G. E., Casper, D. L. D., Gall, C. $ Goodenough, D. A. (1985). Gap junction structures. VII Analysis of connexon images obtained with cationic and anionic negative strains. J. Mol. Biol. 184, 81-98. Barr. L.. Dewey, M. M.t Berger, W. (1965). Propagation of action potentials and the structure of the nexus in cardiac muscle. J. Gen. Physiol. 48, 797-823. Beyer, E. C., Paul, D. L. & Goodenough, D. A. (1987). Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J. CeZZ BioE. 105, 2621-2629. Beyer, E. C., Kistler, J., Paul, D. L. & Goodenough, D. A. (1989). Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108, 595-605. Caspar, D. L. D., Goodenough, D. A., Makowski, I,. & Phillips, W. C. (1977). Gap junction structures. I. Correlated electron microscopy and X-ray diffraction. J. Cell Biol. 74, 605628. De Mello, W. C. (1982). Intercellular communication in cardiac muscle. Circ. Res. 51, l-9. DuPont, E., El Aoumari, A.. Roustiau-Severe, S., Briand, J. P. & Gros. D. (1988). Immunological characterization of rat cardiac gap junctions: presence of common antigenic determinants in heart of other vertebrate species and in various organs. J. Memb. Biol. 104, 119128. El Aoumari, A. E., Fromaget, C., DuPont, E., Reggio, H., Durbec, P., Briand J.-P., Boiler, K., Kreitman, B. & Gros. I). (1990). Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain. J. Memb. Biol. 115, 224-240. Erickson, H. P. & Klug, A. (1971). Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs. Phil. Trans. Roy. See. Land. Ser. B. 261. 105118. Gimlich, R. L., Kumar. N. M. & Gilula, N. B. (1990). Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J. CeEZ Biol. 110, 597-605. Goodenough, D. A.. Paul, D. L. & Jesaitis, L. (1988). Topological distribution of two connexin32 ant,igenia sites in intact and split rodent hepatocyte gap junctions. J. Cell Biol. 107, 1817-1824. Green, N., Alexander, H., Olson, A.. Alexander, S., Shinnick, T. M., Sutcliffe, J. G. & Lerner, R. A. (1982). Immunogenic structure of the influenza virus hemagglutinin. Cell, 28, 477-487. Gras, D. B., Nicholson, B. J. & Revel, ,J.-P. (1983). Comparative analysis of the gap junction protein from rat heart and liver: is there a tissue specificity of gap junctions? Cell, 35, 5399549. Harfst, E., Severs, N. J. t Green, C. R. (1990). Cardiac

Cardiac Gap dun&ion myocyte gap junctions: evidence for a major connexon protein with an apparent molecular mass of 70,000. J. Cell Sci. 96, 591-604. Henderson, R., Baldwin, J. M., Downing, K. H., Lepault, J. & Zemlin, F. (1986). Structure of purple membrane from Halobacterium Halobium: recording, measurement and evaluation of electron micrographs at 3.5 a resolution. Ultramicroscopy, 19, 147-178. Hertzberg, E. L. (1984). A detergent-independent procedure for the isolation of gap junctions from rat liver. J. Biol. Chem. 259, 9936-9943. Hertzberg, E. L., Disher, R. M., Tiller, A. A., Zhou, Y. & Cook. R. G. (1988). Topology of the M, 27,000 liver gap junction protein: cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive. J. Biol. Chem. 263, 19105-19111. Houghten, R. A. (1985). General method for the rapid solid-phase synthesis of large numbers of peptides: Specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Nat. Acod. &i., U.S.A. 82, 5131-5135. Johnson. G. D. & Nogueira Araujo, G. M. de C. (1981). A simple method of reducing the fading of immunomicroscopy. fluorescence during J. Immunol. Methods, 43, 349-350. Kensler, R. W. & Goodenough, D. A. (1980). Isolation of mouse myocardial gap junctions. J. Cell Biol. 86, 755-764. Kistler, .J., Christie, D. & Bullivant, S. (1988). Homologies between gap junction proteins in lens. heart and liver. Nature (London), 331, 721-723. Kumar. N. M. & Gilula. N. B. (1986). Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J. Cell Biol. 103, 767-716. Laemmli. U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. Laird, D. W. & Revel, J-P. (1990). Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes. J. Cell Sci. 97, 109-117. Liu. F.-T., Zinnecker, M., Hamaoka, T. & Katz, D. H. (1979). New procedures for preparation and isolation of conjugat,es of proteins and a synthetic copolymer of o-amino acids and immunochemical characterization of such conjugates. Biochemistry, 18,6%697. Loewenstein. W. R. (1981). ,Junctional intercellular communication: the cell-to-cell membrane channel. Physiol. Rev. 61, 829-913. Makowski, L., Caspar, D. L. D., Phillips, W. C. & Goodenough, D. A. (1977). Gap junction structures. IT. Analysis of the X-ray diffraction data. J. Cell Biol. 74. 62S645. Manjunath. C. K. & Page, E. (1986). Rat heart gap junctions as disulfide-bonded connexon multimers: their depolymerization and solubilization in deoxycholate. J. Memb. Biol. 90, 43-57. Manjunath. C. K.. Goings, G. E. & Page, E. (1984a). Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins. Amer. J. Physiol. 246, H865-H875. Manjunath. C. K., Goings, G. E. & Page, E. (1984b). Detergent sensitivity and splitting of isolated liver gap junctions. J. Memb. Biol. 78, 147-155. Manjunath, C. K., Goings. G. E. & Page, E. (1985). Proteolysis of cardiac gap junctions during their isolat.ion from rat hearts. J. Memb. Biol. 85, 159-168.

Structure

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Yeager, M. & Gilula, N. B. (1999). Membrane topology and quaternary structure of the cardiac gap junction ion channel. J. Amer. Coil. Cardiol. 15, 2A. Zervos, A. S., Hope, J. & Evans, W. H. (1985). Preparation of a gap junction fraction from uteri of pregnant rats: the 2%kD polypeptides of uterus, liver, and heart gap junctions are homologous. J. Cell Biol. 101, 1363-1370. Zhang, J.-T. & Nicholson, B. J. (1989). Sequence and

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tissue distribution of a second protein of hepatic gap junctions, Cx26. as deduced from its cDNA. J. Cell Biol. 109, 3391-3401. Zimmer, D. B.: Green, C. R,., Evans. W. H. & Gilula, N, B. (1987). Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J. Bid. Chem. 262, 7751-7763.

by D. DeRosier