Three Differentially Expressed Na,K-ATPase a Subunit Isoforms: Structural and Functional Implications Victor ia L. M. Her re ra ,* J a n e t R . E m a n u e l , * Nelson Ruiz-Opazo,* Rober t Levenson,~ B e r n a r d o N a d a l - G i n a r d *

* Laboratory of Molecular and Cellular Cardiology, Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, and Departments of Pediatrics, Physiology, and Biophysics, Harvard Medical School, Boston, Massachusetts 02115; and ~Department of CeU Biology, Yale University School of Medicine, New Haven, Connecticut 06510

Abstract. We have characterized cDNAs coding for three Na,K-ATPase tx subunit isoforms from the rat, a species resistant to ouabain. Northern blot and Sl-nu- clease mapping analyses revealed that these a subunit mRNAs are expressed in a tissue-specific and develop- mentally regulated fashion. The mRNA for the a l iso- form, --~-4.5 kb long, is expressed in all fetal and adult rat tissues examined. The a2 mRNA, also ---4.5 kb long, is expressed predominantly in brain and fetal heart. The ix3 cDNA detected two mRNA species: a ---4.5 kb mRNA present in most tissues and a ---6 kb

mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the a sub- units are encoded by a multigene family. Structural analysis of the a subunits from rat and other species predicts a polytopic protein with seven membrane- spanning regions. Isoform diversity of the a subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

T HE plasma membrane protein that directly couples the hydrolysis of ATP to the active transport of Na § and K § across the plasma membrane in most animal cells

is Na,K-ATPase. This transport produces a Na+/K § elec- trochemical gradient to which is coupled net extrusion or ac- cumulation of many other substances against their concen- tration gradients (Kyte, 1981). The Na,K-ATPase, therefore, plays a central role in a variety of physiological processes: regulation of cell volume (MacKnight and l_e.af, 1977), differentiation (Smith et al., 1982), proliferation (Rozengurt and Heppel, 1975), ion/solute uptake in the stomach, intes- tine (Allen and Navran, 1984), liver (Blitzer and Boyer, 1978), kidney (Kyte, 1976a, b), and bone (Baron et al., 1986), propagation of the action potential of muscle and nerve (Thomas, 1972), and modulation of synaptic action (Phillis, 1977). In all tissues from which Na,K-ATPase has been identified it has been shown to consist of two subunits, a and 13. The a subunit is a polypeptide of Mr'~100,000 that con- tains the ATP- and ouabain-binding sites. The a subunit is phosphorylated and undergoes conformational changes dur- ing its reaction cycle (Jorgensen, 1983). The 13 subunit is a glycosylated polypeptide of MRS55,000 whose biochemical function is unknown but appears indispensable for enzy- matic function (Sweadner and Goldin, 1980). Two a subunit

V. L. M. Herrera's and N. Ruiz-Opazo's present address is Section of Molecular Genetics, Cardiovascular Institute, Boston University Medical School, Boston, MA 02118.

isoforms have been identified in the rat (a and a +) (Swead- ner and Gilkeson, 1985; Lytton, 1985) and in brine shrimp (al and a2) (Morohashi and Kawamura, 1984). These pro- tein isoforms differ in SDS polyacrylamide gel mobility and amino-terminal amino acid (aa) ~ sequences. Two antigeni- cally different isoforms with different tissue distribution have also been described in the chicken (Fambrough and Bayne, 1983). The primary structure of the a subunit from three ouabain-sensitive species, sheep kidney (Shull et al., 1985), electric ray electroplax (Kawakami et al., 1985) and pig kid- ney (Ovchinnikov et al., 1986), has been determined from cDNA clones.

The reported sequence conservation of the Na,K-ATPase among species is in marked contrast with the wide variety of functions carried out by this enzyme in different cell types, at various developmental stages, and physiological condi- tions. The present study was undertaken with two main ob- jectives in mind: first, to determine the extent and nature of tissue-specific isoform diversity of the a subunit; and sec- ond, to determine the primary structure of the a subunit(s) from an ouabain-resistant species to better understand struc- ture-function relationships, the molecular mechanism(s) of ion transport, and the basis for differential ouabain sensitiv- ity among species.

We have isolated and characterized cDNAs coding for

1. Abbreviations used in this paper: aa, amino acid; GES, Goldman- Engleman-Steitz scale; H, hydrophobic region.

�9 The Rockefeller University Press, 0021-9525/87/10/1855/11 $2.00 The Journal of Cell Biology, Volume 105, October 1987 1855-1865 1855

three Na,K-ATPase a subunit isoforms from rat, an ouabain- resistant species. Our results suggest that in the rat Na,K- ATPase a subunit is encoded by a multigene family that is expressed in a tissue-specific and developmentally regulated manner. Comparative analysis of the primary and deduced secondary structures of the a subunit protein isoforms from rat and other species predicts a polytopic protein containing seven putative membrane-spanning domains and two puta- tive regions involved in ouabain binding. While the overall structure of the a subunit from rat and other species appears to be conserved, structural differences between rat isoforms were detected. Such differences are consistent with the hy- pothesis that Na,K-ATPase tt subunit isoforms play specific functional roles.

Materials and Methods

Isolation and Characterization of cDNA Clones Rat brain and liver ~.gtll cDNA libraries were plated (50,000-100,000 recombinant plaque-forming units per plate) and transferred to nitrocellu- lose filters (Schleicher & Schuell, Keene, NH). Filters were prepared for hybridization by the method of Benton and Davis (1977). Random hexa- nucleotide-primed 32p-dCTP (Amersham Corp., Arlington Heights, IL)- labeled cDNA (RB5) probe was prepared by the method of Feinberg and Vogelstein (1983) to a specific activity of 109 cpm/l~g DNA, and hybridized to the filters overnight. Filters were washed at varying stringencies, followed by autoradiography. Positive clones were plaque purified and their cDNA inserts characterized by standard restriction endonuclease-mapping proce- dures.

Isolation of mRNA and Northern Blot Analysis

Fetal (18-d gestation) and adult rat tissue mRNAs were isolated from Sprague-Dawley rats by the method of Chirgwin et al. (1979). A panel of fetal and adult tissue total cellular RNA (20 Ixg of RNA per sample) was separated electrophoretically on 1% agarose gels containing formaldehyde as previously described (Lehrach et al., 1977). The RNA was transferred to nitrocellulose filters (Schleicher & Schuell) and hybridized with 0.5-1 x 106 cpm/ml random hexanucleotide-primed 32p-dCTP-labeled probes, at 42~ in 5x SSC (Ix SSC = 150 mM NaC1, 50 mM sodium citrate), 50% formamide (Thomas, 1980). Washes were performed at 65~ in 0.1x SSC, 0.1% SDS for 1 h.

S1-Nuclease Mapping Analysis End-labeled, double-strand cDNA restriction fragment probes (Fig. 3 C) were prepared either by kinase (New England Biolabs, Beverly, MA) label- ing with 32P-ATP (5'-end labeled probes) or with 32P-ddATP (Amersham Corp.) using terminal transferase (New England Biolabs) (T-end-labeled probes). Double-strand end-labeled probes were hybridized to 20 ~tg of total cellular RNA under RNA-looping conditions as described previously (Casey and Davidson, 1977; Berk and Sharp, 1977). The reaction mixture was incubated with 200 Ixm of Sl-nuclease (Boehringer Mannheim Bio- chemicals, Indianapolis, IN) at 25~ for 1 h. Sl-nuclease-resistant products were ethanol precipitated and size separated on 8% polyacrylamide gels. Negative controls using tRNA were run to assess for any reannealing of the end-labeled probes.

Southern Blot Analysis Rat liver genomic DNA was isolated by a modification of the method de- scribed by Blin and Stafford (1976). Oenomic DNA was digested with a panel of restriction endonucleases and the DNA fragments separated elec- tropboretically on 1% agarose gels. The DNA was transferred to Zeta-bind filters (AMF Cuno Precision Control Products, Meriden, CT) by the method of Southern (1975). Hybridization was carried out with cDNA probes specific for each a subunit isoform.

DNA Sequence Analysis DNA sequence analysis was performed by the dideoxy chain-termination method of Sanger et al. (1977) using 35S-dATP (Amersham Corp) as radio-

o~-1 i 0.5kb

~l

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- �9 : . �9

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~ - 3 " - - L _ " - - t _ . . _ . I I - - ' ~ 11--"

ES Pv Pv Pv B KS E

R ~ 3 - a (2 .7kb) I i

R o ' 3 - b (2 .5kb ) I

Figure 1. Restriction map analysis and sequencing strategy of three classes of cDNA clones of the Na,K-ATPase u subunit. Na,K- ATPase ct subunit cDNAs were classified into three groups (al, a2, and a3), according to restriction maps generated by digestion with different restriction endonucleases: B, Bam HI; E, Eco RI; K, Kpn; P, Pst I; Pv, Pvu I; R, Rsa I; X, Xho I; K, Kpn I; S, Sac I; X, Xba I. Restriction fragments were subcloned into MI3 vectors and the nucleotide sequence obtained. (o--,) Origin and direction of nucleo- tide sequencing; (m-~) synthetic oligonucleotide-primed nucleotide sequence. The cDNA clones are aligned according to their deduced amino acid sequences.

active label. Appropriate restriction fragments were isolated from low melt agarose gels and subeloned directly into the corresponding MI3 vectors (mpl8 and/or mp19) (Messing, 1983). Single-strand DNA templates were isolated and subjected to nucleotide sequence analysis (Bethesda Research Laboratories, Gaithersburg, MD). An average of 300-350 nucleotide se- quences was obtained. A significant amount of the sequence was obtained using template-specific 17-mer primers synthesized on a DNA synthesizer (Biosearch, San Rafael, CA) based on the known DNA sequence. Each re- gion was sequenced at least twice to reduce ambiguities and confirm over- lapping regions. Overlapping regions of respective eDNA clones were se- quenced multiple times in both orientations.

Results

Isolation of Three Classes of Na,K-ATPase a Subunit eDNA Clones To search for Na,K-ATPase a subunit isoforms from an ouabain-resistant species, several rat cDNA libraries were screened using the previously characterized RB5 eDNA clone (Schneider et al., 1985) as probe. Several clones were isolated. Ral-a, Rct2-a, Rct2-d, Rct3-a, and Ra3-b were iso- lated from a random-primed adult rat brain ~,gtll eDNA li- brary. Another clone, Red-b, was isolated from an adult rat liver ggtll eDNA library. Restriction map analysis identified three classes ofcDNA clones (Fig. 1): class 1 (al) consisted of Ral-a and Ral-b; class 2 (ct2) consisted of Ra2-a and d; while class 3 (ct3) consisted of Rct3-a and Ra3-b. Clones representing 3' extensions of a2, Ra2-b and c were subse-

The Journal of Cell Biology, Volume 105, 1987 1856

quently isolated from an oligo dT-pr imed adult rat brain cDNA library using the 3'-most 400-bp Pst I/Eco RI restric-

tion fragment of Ra2-a as probe (Fig. 1). Many other a2 clones were isolated from several libraries but did not extend further toward the 3' UT presumably because of the presence of internal Eco RI sites which were not methylated during the cDNA cloning procedure.

Three a Subunit mRNAs, Each Encoded by a Different Gene, Are Expressed in a 1issue-specific and Developmentally Regulated Fashion

The cDNA clones shown in Fig. 1 were used to investigate the tissue distribution and size of each ct subunit mRNA iso- form. Probes were prepared from the cDNA clones spanning the phosphorylation and ATP-binding sites from each class: ctl (Ral-b), a2 (Rct2-a), and Q3 (Ra3-a) (Fig. 1). Each probe was hybridized to separate but identical Northern blots containing equivalent amounts (20 Ixg) of fetal (18-d gesta- tion) and adult rat tissue total cellular RNA. At the same stringent conditions, strikingly different patterns of mRNA distribution were detected (Fig. 2). As shown in Fig. 2 B, the ctl probe hybridized to a =4.5-kb mRNA present in varying

Figure 3. Sl-nuclease mapping analysis of Na,K-ATPase ~tl mRNA isoform. (A) Sl-nuclease protection of the 190-bp-long end-labeled Kpn/Eco RI fragment of clone Rctl-b shown as probe A in C by RNA isolated from different rat fetal (f) and adult (a) tissues. The full-length protected fragment is indicated by a solid arrow. The un- digested probe is shown in the first lane. End-labeled Hae III- digested q~X174 markers are shown in the second lane in base pairs. (B) Sl-nuclease protection pattern of the 3' portion of clone Ral-b extending from the Kpn site into the plasmid vector and shown as probe B in C. The fully protected fragment is indicated by a solid arrow. Sm. lntest, and Sk. M., small intestine and skeletal muscle, respectively. The first lane shows the double-stranded labeled probe. End-labeled Hind III-digested L DNA markers in the sec- ond lane are in base pairs. (C) Map of the different end-labeled re- striction fragments used for Sl-nuclease mapping experiments with *-* indicating the labeling site and its 5' or 3' position. These probes span the Na,K-ATPase u subunit phosphorylation site (P*); FITC- binding site; stop codon (COOH); and poly-A tail (AAAA). ( ~ ) Vector sequences.

Figure 2. Northern blot analyses of Na,K-ATPase ct subunit iso- forms. Three separate but identical Northern blots of equivalent amounts (20 lag) of rat adult (a) and fetal ( f ) tissues were hybrid- ized to a cDNA probe for each Na,K-ATPase a subunit isoform spanning comparable regions. (A) Ethidium bromide-stained pic- ture of the gels before transfer. (B, C, and D) The pattern of hybrid- ization to al (codons 402-938), a2 (5'UT to codon 940), and ct3 (codon 44 to the 3' end) cDNA clones, respectively. (Arrow) The hybridizing mRNA species. Size of ribosomal RNA subunits (28S, 18S, 5S) are noted on the left. Sk. M., skeletal muscle. Longer ex- posure of the Northern blot in D (not shown) reveals a ~4.5-kb mRNA hybridizing band in fetal liver and skeletal muscle.

amounts in all tissues examined. Different patterns of expres- sion are also detected during development. The ctl mRNA is more abundant in fetal than adult kidney, whereas it is more abundant in adult than fetal brain. An almost equal level of al mRNA is detected in fetal and adult heart, as well as in fetal and adult skeletal muscle. As shown in Fig. 2 C, the ct2 probe also detects a -4 .5 -kb mRNA but this species is present only in brain and fetal heart. Like the a l mRNA, the ct2 isoform is also more abundant in adult than fetal brain. As shown in Fig. 2 D, the ct3 cDNA probe detects two mRNAs of different sizes, ~=6 and ~4.5 kb. The ~6-kb mRNA is detected only in fetal brain and adult brain, heart,

Herrera et al. Na,K-ATPase a Subunit lsoforms 1857

Figure 4. Southern blot analysis of rat liver DNA. (al) Southern blot of rat liver DNA digested with different restriction endonucleases (Ava I, Barn HI, Eco RI, Nco L Pst I), hybridized to an al isoform-specific DNA probe spanning the 5q.lT to the 3TIT. (a2) Subsequent hybridization of the same blot with an a2 isoform-specific DNA probe spanning from 5'UT to aa 940 (al numbering.) (a3) Subse- quent hybridization of the same South- ern blot with an a3 isoform- specific DNA probe spanning aa 44 to 3q_rl" (al numbering). Hae III- and Hind III-digested phage DNA markers are on the left in kilobase pairs.

and skeletal muscle. The lower hybridizing band (~4.5 kb) is similar in size to the al and a2 mRNAs. However, it is unlikely that this band represents hybridization to al and/or a2 mRNAs since the relative abundance of the mRNAs that are detected by the a3 probe in individual tissues is strikingly different. Whether the two mRNAs that are detected by the a3 probe are the products of the same or different and highly homologous genes remains to be determined. The smaller mRNA species in adult heart, skeletal muscle, and fetal liver have a slightly faster mobility than in other tissues. It has not yet been determined whether the tissues that exhibit this mo- bility difference express yet another isoform, although con- sidering the stringency of hybridization, this is unlikely.

To confirm that the al cDNA probe truly detected al mRNA and not other cross-hybridizing isoforms mRNAs, Sl-nuclease mapping analysis was performed using different regions of the entire RB5 and Ral-b eDNA clones (Fig. 3 C). The results of two representative experiments are shown in Fig. 3, A and B. Full protection of each end-labeled probe used, spanning different regions of the mRNA, was detected with the mRNA from all fetal and adult tissues studied. Par- tially protected fragments were also observed that varied in intensity in proportion to the fully protected fragments. These bands were not reproducible in other Sl-nuclease mapping experiments using end-labeled probes of different sizes in the 5' and 3' direction and probably represent ar- tifacts of Sl-nuclease digestion. These results confirm that the al mRNA is expressed in every tissue examined and that the ctl mRNA detected by the al eDNA probe is not the re- sult of cross-hybridization with other a subunit mRNAs.

To investigate the genomic complexity of Na,K-ATPase a subunit, rat genomic DNA fragments were hybridized with eDNA probes for each of the a subunits spanning compara- ble regions of each isoform: al (5'-3'UT), a2 (5'UT-codon 940 [al numbering]), and ix3 (codon 44-3'UT [al number- ing]). As shown in Fig. 4, each a subunit probe hybridized to a unique pattern of DNA restriction fragments. This result suggests that each a subunit isoform is encoded by a different

gene. Low stringency hybridization of the genomic blot shown in Fig. 4 detects additional hybridizing bands for each of the probes, suggesting the existence of additional genomic sequences that are homologous but not identical to any of the three cDNAs reported here (data not shown).

Comparative Analysis of the Primary Structure of the Na,K-ATPase a Subunit lsoforras

Nucleotide sequences were obtained for all al and a2 eDNA clones, confirming the alignment shown in Fig. 1 by the exis- tence of overlapping regions with identical sequences ob- tained in both orientations. Partial nucleotide sequences have been obtained for a3. Comparison of nucleotide sequences (Fig. 5) show al and ct2 to be 74% homologous with 66% of the differences being at the wobble position. Partial nucleotide sequence confirmed that a3 represents a third a subunit isoform differing from al and ct2 in codon usage and deduced amino acid sequence (data not shown).

Analysis of the deduced aa sequences of the rat a subunit isoforms (Fig. 5) and previously characterized a subunits shows that al from the rat is almost identical (97 % homol- ogy) with the a subunit polypeptides from sheep (Shull et al., 1985) and pig kidney (Ovchinnikov et al., 1986). Sur- prisingly, the rat a2 isoform is only 82-85 % homologous with rat al, and the previously characterized a-subunits from sheep kidney (Shull et al., 1985), pig kidney (Ovchin- nikov et al., 1986), and electric ray electroplax (Kawakami et al., 1985). The amino terminus of a2 differs markedly from other identified sequences (Fig. 6) including the previ- ously described rat brain isoform, a + (Lytton, 1985). It is shorter than al by 10 aa and lacks histidine 13, like the brine shrimp a subunit amino terminus (Morohashi and Kawa- mura, 1984). The lysine rich regions are highly conserved, however, among all the amino termini characterized to date. Interestingly, comparison of the protein- (Hopkins et al., 1976; Cantley, 1981; Collins et al., 1983; Morohashi and Kawamura, 1984) and cDNA-derived al-type aa sequences

The Journal of Cell Biology, Volume 105, 1987 1858

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D - - - "I - - -

K T S A T I r A V L $ R [ A G L C N R V A u F q A N Q E N L V P I L K R A V A G O T A S E S A L L K C I r E u 4 6 2 RC%; TC.It ~ ~ ~ TTC ICT Cu TC~: ~ A ~ ICT C~T CTC T~T AP,C A(}C; GCA GT~ TTT CA{:; ICT ~ CA(; CAP. AAC CTG CCT P.TC CTG PAC, CC, T G(~ ~T~. ~ ~ CAT ICT TCC GAG TCC, GCE, ~TC CTA AAG ~ ATC C,P,G ~'TC 1640

. . . . GC . . . CA . . . . . . . G.G . . C . . . . . . CAC . .C . . C . . . . . . . . C . . . C . . . . T . . C . . . A . . . I C GGG . . . . . . . . . . T A.C . . . G .A . .C . . . A.G .~C . .O . . . . . T . . . . . C . .A . . . . . C . . C . .G . .T . . . . . . . . . . . . C.G

- S - H - - V - - - H . . . . . . . . . . K C G - [ } - I - V - - - O - - I . . . . . . . . . F I - T C - ~ - - L

~T~ G ~ ATT CCT TTC AAC TCC .eO~C P.9.C A~G TAC CP, E, CTC TCC ATT CAC AP, C, ~ ~ PAC I C A 3UG ~ CC~[ ~ ~ CTG CTA gTG AT~ P,A{; ~ (]L'X~ ~ ~ ] 796

i l r ~ T~'T T ~ GTG ATG ~ ATG ~GG GAG ~ T ~ AEC P ~ ATA .C. .C(; . . . . . . . A .A. CT . . . . C.C . .A ~ A . . . A G . .A Gl'C .CC . . . . . . . . C . . . . . . . . . . . T . . . . . A . . . . . . . . A . . . . . C . .T G . . . C T C~C~G ~ . . C C PAT . .C ~ CC.C~ T . . . . T . . . . . . . . . . . . . . . . . . . T . . .

S S - - K L - - - R N K - V A . . . . . . . . . . . . . . . E T E O P N O N R V . . . . . . . . .

R I L O R C S S ~ I L L H G K E q P L ~ D E E L K D A F Q N ~ A Y L E L G G L G E r R V L G F C H L L L ~ P O 5 6 2

~TC CII~ ~ ~ TE;C AGT TCT ATC CTC CTC ~ GGC AP,C- ~ ~ CCC CTG ~ CJLA ~ CTG Cu~.C C, AC I;CC TTT CAG PAT ICC TP.E CTG ~ CTG GGT GGC CTG ~ C~%A CGT GTG CTA ~GT X'fC T~C C..e,C CTC CTT CTG CCT GAC [846

C.C . . T . . . . . . . . C . . T GCG A.C . . . . . . . . G . .G . . . . . . . . . . . . . . T . . . . . T . .G � 9 1 4 9 1 4 9 R . . . . . . . G . . . . . C . . . . . C . . . . . . . . . . . . . . T . . . . . . . . . . . C . -G . . . . . . . . G . . . . . . . . . . . T TA. TN~ . . . . . G . .G

. . . . . . A T - - - q . . . . . . . . . 14 - E . . . . . . . . . . . . . . . . . . . . . Y v - - E

E Q F P E G r Q ~ F D T O E V N F P v V o N L C F V G L [ S V N I O P P R A A V P ~ O A V G K C R S A G V T K 6 1 2

C.e,G TTr CCT C.{d~ GGC "r~C CA~ TTT ~ PL"T EAT ~ GTC PAT TTC CCC GTG CAT AP,C: ETC TIC TTC GTG GGT CTT J~TC TEE P.TC ATT ~ CCI CCT ~ ICT ICT GTE C'C'C. GAT ICT GT~ Gs AAA ~ C~C E (;CT ~ ATT ~ 1996

. . . . . . . . C . .C A.G . . . . . T GCs . . . . . . TG . . . . . . C . .G . .E . . . A.. ACA . .C . . . . . T . . . . . . . . . . . . . . C . .G . . . . . . . . C . . . . . . . . C . .G . .A . . . . . . . . T . . . . . . . . T . . . . . . . . . . . . . . T . .A . .C . .C . . .

E - - - ~ - - A - - C - O - - - T T . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . .

V I I I v T G O H V P [ T A K A I A K G e V G I I S E G N E T I F V E O ! A ~ R L 14 T W P V N Q V N P R 0 A q ' K A 6 8 2

G'TC ATE RTG ~ ~ ~ ~ CAT C~#. ATC ~ GLr'( ' ~ ~ ATT GCT PAG (~G(; ~TC (~GC ATT ATC TCA ~ G~;T E C~PG J&CC GTG ~ GAC AT~ ICT GCC C~C CTC PAC ATT CCA G'rG E CAG C.TG CV~C CL'C ~ CAT GCC APs ~ 2146

. . . . . . . . . . . . . . C . . C . � 9 �9149 . �9 . . . . . T . . G . , G . . . . . C . . C . , A �9 .T A ~ . . . . . C . . . . . C . .G . . . . . . . . . . . T . . . . . G . . . . . E . . . . . . . . ~ . . . . . . . . C � 9 1 4 9 . .C .G . . . . . . C . . . . . . . . G . . . . . . . . A �9 . � 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . .

F S B A / C V V H G ~ O L ~ K O N T S E E L O O ~ I L R Y H T E 1 V F ~ R T S P Q Q K L I V l V s G C Q R Q G ~ V l v 7 ) 2

T~ . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ ~PA . . . . . . . . . . ~ . . . . . . ~ ~ ~ ~ ~ ~ . . . . . . 2298 . . . . . G A . T . . . . . . . C . . . . C.C . . . . . . T .C . . . . . . . . . C . . A . T . .C . .C . .C C.C . A . A . . . . . . . T . . . . . C . . . . . . . . C C.A . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . C . T A .A . . . . . A . . A . . . . T . . .

- - I - - T . . . . F - - - ~ I - E - - q N . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A u T G O G V N ~ O $ P A L K K A O I V G Y ~ 14 G I V G S O V V $ K q A A D M I L V L O O N F A $ T V T V G u 7 6 2

GL'T GTC ~ GGG C.P.T GGT GTC PAT G~C TCT CCA OCT TTG ~ ~ ~ CAT ATT GGG GTT ~ ~TG ~ ATT GTT GC4~ TC'~ CAT GTG T~'C AAG CPA ICT C.CT CAC ATG ATT CTT CTG GAT CA(:: ~ TTI OCC TCC ATC GTG ACT ~ GTA 2446

. . . . . (; , .T ..C . . . . . . . . G . . . . . . . . C . .T . . . C . . . . G . . . . . I; . . . . . . . . . . . G . . . . . . . . C . . . . C . . . . . . T . . . . . C . .T . . . . . G . . . . . C . . . . . . . . . . . G . . . . . . . . . . . T . . . . . I" . . . . . T ..C . . . . . T , .C

. . . . . . . . . . . . . . . . . . . . . . . . . - - ~ - - - - - - H ; - [ -- - -- . . . . . . . . ,:,-OOA--R - E ( G R L ] F" O ~ l l L K K $ [ A Y T L I T ~ N I P E ~ u P F ~ F L I F [ ! A N ! P L ~ F P L G T u T 1 L ~ I V O L 8 1 2

C41,A ~ GGT C:GT CTG ATA ITT C,.~T ~ TTC ~ ~ I'CI~ ATT ~CI TAC ~ CTA AC e. PGT P, AC A]T CCC~ ~ ATC ACE CCC TTC TTG AlrP~ TTT ATT ATT ~ A.%C ATT CCA CTG CCC CTG GGC ACC GTG ~ ATC CTC TIC ATr ~ l~rG 2596

. . ~ . . . . . C . .C . . . . . C . . . . . C . . . C . . . . . . . . . . . . . C . .C . . . . . T . .G . .C . .C . . . . . C . .T . .G . . . . . A . . . . . . C . T CTC . .U . .C . . . . . . . . . . . . . . . . . . . . . G . . C . C T . . . . . . A.C . . . . . . . . . . . . . . . . . . C . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . L - - W ~ . . . . . . . . I . . . . . . . I H e

G T O N V P A I I S L ~ Y E Q A ( S D ~ I M K R Q P R N P K ~ T D K L V N ( R L I WFS N A Y G Q I G M I V Q A 862

ACT ~ ATG G'n" ~ ~ ATe TCT ~ GEC TAT ~ ~ ICT ~ AGT ~ ATC P, TG ~ ~G ~ CCC KC4~ PAT CCC .~ ~C~ C9~C ~ CT~ GT~ ~ ~ CGT CTG ATC ~ ATG GL'C TAT ~ ~ ~.TC GGT ATG ATC C~G GICE 2746

. . . . . C . . . . . . . ~ . ,T A .A . . . . . C "r . . . . . . . c . . G O C T . . A . .G . . . . . . . . . . . . . . . . . . . . . . . . . . G . .C . .A ~ . .A . . . . . . . . G . . C . . . . . A A . G . . C . . . . . . . . . . . . . . . . . G . . . . . . . . G . . . . . . . . . . . .

. . . . D - T . . . . . . A . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . .

I L G G F F T Y e ~ u I L A E N G r L P V F H L L G I R E T I f D O R �9 I 14 O V E O W S Y G Q Q �9 T Y E Q W R K 9 1 2

CT~ ~ ~ : 1'IC TTC ACT TAT TTT GTG AT[ CTG GET ~ A,elC GGI ITC C'I~; r TTr CJ&C CTG TTG GGC ATC CC.A ~ ACC TGI; C.~.T C.,o,C C(:-C TGG AT(: PAT CAT GTC- GAG GAC AIC T~C GGG CAG ~ TGG ~CC TAT ~ ~ ~ ~ 2896

�9 .C . . . . . T . . . . . . T.C . .C . . . . . C ..C . . . . . A . .A . .T . . . . . . T . . . . . ( ~ . A . . . . . G . . . . . . . . . . G CTC .A . . . . . . . . . . . . . A(~T G . . . . C . .(: C.. , .A . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . .

. . . . . 5 . . . . . . . . . . . . G N - V - - - L N . . . . T V - - L . . . . . . . . . . . . . .

I H7 I | V E F T C H T V A F I r u S I u V u Q ~ I A O L V I C I ( T R V R N S V F Q Q G M K V N K I L | F G L F E ~ E T 9 6 2

ATT GTG EJ~ TTC ~ T~C CA{: N~G GCC TTC TTT GTC AGT ATC GTG GTA GTG CAr., ~GG I C T GAC I T G GTC AT(: T I C N~G ~CC ~ ~ G PAT TCC GTC TrC C ~ ~ CC~ ATG ~ A~C ~ ATC TTA ATA T R GGC CTC TTT CJ~A EAG ~ A 3 0 4 6

G.C , . T . . . . . . . . C . . . . . . . . A . . . . . . . . C . . G . . C . . A . . . . . ~ . . C . . . . . . . . . . . . C . . A . . . . l . . . . . . . . . . . G

�9 . . . . . . . . . . . . . . . . . . . . . I . . . . .

A L A A F L $ Y ~ C P G 14 G A A L R N ~ Y P L I ( p T �9 �9 F C ~ F P Y $ L L 1 F V ~ Y O E u R I ( L I | R ~ R R 1 0 1 2

ICT CTT GL-T G{;T IT(: CTG TCC TAC TIC OCT ~ AT(; ~ T ~ GClC CIT AGC, ATG TAT ~ CTC ~ CCT ~ TGG TGG TTC TGT GL'(: I~{C ~ TAC TCC CTT CTC AT(: TTC GTG TAT CA(; Ca~C, GTG ~ ~ CTC ATC ATC ~ ~ ~ 3196

P G G �9 V E K E �9 I �9 Y 1 0 2 3

CI~T ~ GGC TGG GTG GN; ~qG GAA ~CC T N : T#,C T N 3 L ` C C A ~ T G C C C T ( ~ T G ~ T T G T ~ C C A ~ T G ~ A ~ C T ~ C ( ~ T r ~ T T T G T G T A C T T C A p ` G T C T T ( ~ A ~ C T C ~ T C T p ` C : C ~ T G G T ~ T ~ T ~ T ~ T ~ 3 ] 8 4

CllAluG T ~ I T GI~ T T GCA~ I T Ct ~ TT GTIN~ T T A~{.#,C T AT (~GCA~k(:a(kA T C T Cp~GEEAIkGGA T GT CA T ~ T ~ , C I T ~T~3~ I~C.e~AAT G~d~ T D~I~ T A /~A T T TT GGC T ~ & ~ _ & n &&~pp t, e ~ A A j ~ A j ~ 3 ~ 3

Figure 5. Nucleotide and deduced amino acid sequence homology between N a , K - A T P a s e o . subunit isoforms ~tl and o.2. The al nt sequences (fine print, numbered) and aa sequences expressed in one letter code (bold print, numbered) are presented. The ~2 aa (bold print, unnum- bered) and nucleotide sequences (fine print, unnumbered) are aligned below. Spaces mark shifts in either sequence to allow optimal align- ment for aa insertions/deletions. �9 marks every 10 aa in ~tl. (. and -) Nucleotide and aa homology between (tl and ~t2 isoforms, respec- tively. Hydrophobic putative membrane-spanning regions I--It-[-[7 are indicated; 1- and 2-OUA-R, putative ouabain-binding regions; P*, pbosphorylation site; FSB, I, 5'-(p-tluorosulfonyl) benzoyladenosine-binding sites; F/TO, fluorescein 5'-isothiocyanate-binding site; I, the 10-aa-long region of nonhomology.

Herrera et al. Na,K-ATPase ct Subunit lsoforms 1859

RAT a +

RAT (~1

S.K. cDNA

P.K. cDNA

E.R.E. cDNA

RAT "2

B.S.c~ 1

B.S.c~ 2

E E

MGKGV GRY[~EPAAVSEHC~--<KAKKERDMDELKKEVSMDDHKLS 1

i,o,ov RRLKKD[IKSSF I(-~ ] KI~ tKKEV~ i i l I I "E~ I~S

Figure 6. Comparison of amino-terminal sequences of different Na,K-ATPase ct subunits. Amino- terminal sequences of different ct subunit isoforms are aligned. The different ct subunits presented are: RATa+ (Lytton, 1985); RATer1 and ~t2 (this paper); S. K. cDNA, sheep kidney ct subunit (Shull et al., 1985); P. K. cDNA, pig kidney ~t subunit (Ovchinnikov et al., 1986); E. R. E. cDNA, elec- tric ray electroplax ct subunit (Kawakami et al., 1985); B. S. al and a2, brine shrimp al and ct2 isoforms, respectively (Morohashi et al., 1984). The homologous regions among the different se- quences have been boxed. Sequences have been aligned and gaps (-) introduced to maximize ho- mology. The five cDNA-derived aa that are absent from the respective characterized mature protein are segregated from the remainder of the se- quences.

(Shull et al., 1985; Kawakami et al., 1985; Ovchinnikov et al., 1986; this paper) reveals the presence of a highly con- served five amino acid-long amino-terminal peptide that has not been detected in the mature protein. This peptide does not have the characteristics of a signal sequence (Kriel, 1981) and suggests the existence of a novel and so far undescribed posttranslational processing. Furthermore, the a2 isoform is also 10 aa longer than the brine shrimp a l and a2 subunits, raising the possibility of a similar amino-terminal processing event for this isoform. The nature and significance of these posttranslational modifications remain to be elucidated.

The peptides identified for certain functional domains and all the putative membrane-spanning regions are all highly conserved at the aa level in the different a subunit cDNAs characterized to date, including the rat isoforms (Fig. 5). These regions include the phosphorylation site, CSDK (Bas- tide et al., 1973); putative ouabain-binding site, YTWLE (Shull et al., 1985); and two ATP analogue-binding sites, FITC (HLLUMKGAPER [Farley et al., 1984]) and 5 ' - (p-

I , FITC-site

Na, K-ATPase ~ o ~ " ~ 0~1 E I PF N S T N K Y Q L S I H K N P N A S E P K H L L V M K G A P E R I L D R C

o o o o ( J - - -

~2 Gee |174 i �9 " ~ �9 " ~ . . . . . " ~ * " E T E D P N D N R Y " ~ ~ ~ ~ ~ ~ ~ ~ . . . . . .

O r OQ O ( I I . . . . .

Ca-ATPase S low F S R D R K S M S V Y C T P N K P S R T S M S K M F V K G A P E G V I DRC

Fast . . . . . . . . . . . . s . A - s . " A A V G N . . . . . . . . . . . . . . .

Figure 7. Regions of nonhomology in the Na,K-ATPase a subunit isoforms r and a2. Comparison with Ca-ATPase isoforms. A 10- aa-long region of nonhomology (I) is noted between al and ct2 lo- cated in proximity to the FITC-binding site. In this region differ- ences in charged aa composition ( + / - ) and in predicted secondary structure, a-helix (*), ~sheet (-), and turn (vw,) by the Chou and Fasman (1978) algorithm are noted. A similar region of nonhomol- ogy also exists in the slow and fast twitch rabbit muscle isoforms of the Ca-ATPase (Brandl et al., 1986).

fluorosulfonyl) benzoyladenosine (FSBA)-binding sites ([a] MTVAHMWFDNOIHEADT, and [b] DISHENLDDILH- YHTEIVF [Ohta et al., 1985]).

A region of significant nonhomology between a l and a2 isoforms was found in proximity to the ATP analogue (FITC)-binding site. This region (1) is located 5 aa upstream to the lysine residue identified as the FITC-binding site (Fig. 7). It spans 10 consecutive aa and differences in net charge, predicted secondary structure (Chou and Fasman, 1978), and hydrophilicity (Kyte and Doolittle, 1982) are noted between a l and a2. Interestingly, a similar region of nonhomology is noted in the two Ca-ATPase isoforms (Brandl et al., 1986), also in proximity to the FITC-binding site (Fig. 7). 2

Na, K-ATPase a Subuni t Topography

To gain some insight into the rat a l and a2 subunit topogra- phy, we analyzed the aa sequences to determine the putative hydrophobic membrane-spanning regions. Hydropathy plots of a l and a2 were obtained using the Kyte-Doolittle (Kyte and Doolittle, 1982) and the Goldman-Engleman-Steitz (GES) (Engelman et al., 1986) scales. The GES hydropathy plots were obtained using a window averaging of 20 aa since this is the average length required for an a-helix to cross the plasma membrane lipid bilayer (Engelman et al., 1986). A 14-aa window averaging was also applied because the mini- mum number of aa to span the plasma membrane has been experimentally shown to be 12-14 aa (Adams and Rose, 1985). Comparative analysis of the different hydropathy plots identify seven putative membrane-spanning regions. Five regions, Hi and H3-6 (Fig. 8), fulfill the GES criteria for membrane-spanning regions (Engelman et al., 1986). The minimum total free energy of transfer to water for a 12-aa membrane-spanning region was calculated to be 20 kcal/mol from the GES hydropathy plot of an experimentally proven transmembrane region of 12 aa (Adams and Rose, 1985; En-

2. While this paper was under review, Shull et al. (1986) reported the cDNA sequences for three a-subunit isoforms. Although there are scattered nucleotide differences, the three a subunit isoforms reported here most likely correspond to the ones reported by Shull et al. (1986). The differences in length and sequence of the 3'UT of the al isoform are unaccounted for at this time.

The Journal of Cell Biology, Volume 105, 1987 1860

2

1

0

-1

- 2

- 3

- 4

- 5 (2)

(1)

(0)

( -1)

( -2 )

( -3 ]

( -4 )

( -5 )

(X-

, - o ~ . - . , ~ F.To 2 - 0 0 . - .

I1 I IOI I".t A ~ FSBA ~ , I ~ /1 FSSA I '1 P'I I ! v ~ ~ l i ~ I r'a. t t J~ m / t

'-~'~1-" | . . c 2-oo.-,

/1 / 121 l / 3 4 ~/I FSBA ~ / ~ FSBA 1

I H~ H 2 H a H 4 H S H 6 H r (el 1 16)

" I E " 1 E " ' I m " I m ~ E . E . ~ E . ~ E , . , e , '1911 I I ~1 20129.4 13118.3 20las.2 28129.4 27131.4 25 28.8 14 16.5, , 5.3,12,13.r

~.2 ~ol23.5 , e l l r . e 2114o.3 2e12g.2 2r13,.r 251~.~ '~1'~-~ ' '

0 100 200 (aa) (aa)

Figure 8. Hydropathy plots ofNa, K-ATPase a subunit isoforms, at and a2. Hydropathy plots of the rat al and ct2 polypeptides are presented using the GES scale. The vertical axis marks the free energy of transfer to water per amino acid averaged over 14 aa, and the horizontal axis, the unit amino acid. Putative hydrophobic membrane-spanning domains are noted 1-7. Previously identified functional domains are noted for reference: 1- and 2-OUA-R, putative ouabain-binding regions; W, tryptophan; C, cysteine; P*, phosphorylation site; D, aspartic acid; K, lysine; F/TU, fluorescein 5'-isothiocyanate-binding site; FSBA, 5'-(p-fluorosulfonyl) benzoyladenosine-binding sites. The 10-aa- long region of nonhomology (I) is also noted. (Inset) Number of aa residues (aa) per hydrophobic region (HI-H7) and total free energy of transfer to water in kcal/mol (E) for the al and a2 subunits. Weakly hydrophobic regions (8 and 9) in al are also presented with their corresponding aa number (aa) and total free energy of transfer to water in kcal/mol (E).

gelman et al., 1986). Applying these criteria, two regions, H2 and nT, are ambiguous. However, considering previous biochemical data on proteolytic digestion and functional do- main mapping, H2 unequivocally spans the lipid bilayer (Jorgensen et al., 1982a). Therefore, GES 14-aa window- averaging hydropathy plots are presented (Fig. 8) because the membrane-spanning region 2 (H2) in al is predicted to be only 13 aa long with a total free energy of transfer to water of 18.3 kcal/mol. The existence of H2 as a true membrane- spanning region supports the existence of the putative sev- enth transmembrane region, H7. H7 spans 14 aa with a total free energy of transfer to water of 16.5 kcal/mol. The pres- ence of a seventh membrane-spanning domain, H7, places the carboxyl terminus on the extracellular side of the plasma membrane. (Fig. 9 A). If H7 does not span the membrane, however, the carboxyl terminus would be located on the cyto- plasmic side of the plasma membrane. Hydrophobic regions 8 and 9 are unlikely to traverse the membrane because of their low degree of bydrophobicity (Fig. 8). This conclusion is further supported by the observation that the correspond- ing regions of the electric ray electroplax a subunit have an even lower degree of hydrophobicity.

Stabilization of H2 and H7 could be achieved by the for- mation of an aqueous pore by the transmembrane regions (Engelman et al., 1986). Helical wheel analysis (Schiffer and Edmunson, 1967) of HI-H7 identifies a possible relatively hydrophilic pore (Fig. 9 B) that contains charged aa residues.

Discussion

Na,K-ATPase Isoform Diversity Generated by a 1Issue-specific and Developmentally Regulated Multigene Family

Three isoforms of Na,K-ATPase a subunit, al, a2, and a3, have been unambiguously identified by the isolation of three classes of cDNA clones. The respective mRNAs have dis- tinct nucleotide and deduced aa sequences, as well as charac- teristic differential patterns of expression. The two mRNA species ---6.0- and ~4.5-kb mRNAs detected by the a3 cDNA at stringent conditions of hybridization most likely represent transcriptional products of the same gene. Differential utili- zation of polyadenylation signals could account for their size difference as has been observed in other genes (Setzer et al., 1980; Parnes et al., 1983; Capetenaki et al., 1983; Carroll et al., 1986). The significance of the observed tissue-specific variation of mRNA isoforms detected by the a3 cDNA clone remains to be elucidated. The existence of two protein iso- forms produced by alternative splicing is unlikely but cannot be formally excluded at present. Other isoforms in addition to the three presented here most likely exist. This conclusion is supported by the preliminary characterization of several rat genomic clones which differ in primary sequence from the isoforms presented here (unpublished data).

Our results suggest that the three rat a subunit isoforms presented here are encoded by three distinct genes. This view

Herre ra et al. Na,K-ATPase ct Subunit lsoforms 1861

A

EXTRA- CELLULAR

COOH ~.-.aa 1023

',",I =,930 I I I(~1 aa930

N S I

I N T R A - C E L L U L A R aa7"46

J

'..COOH aa1023

B H7 (14aa) (2~ ~ H6

~ 7\ 3 )(25aa)

H5

(20aa) H4 (28aa)

Figure 9. Structural diagram of the Na,K-ATPase r subunit topography. The putative topography of the Na,K-ATPase r subunit is deduced from a detailed analysis of r isoform's hydropathy plot, predicted secondary structure, and helical wheel analysis of the putative membrane- spanning regions. (A) Putative membrane spanning regions HI-H7 delineate the extracellular and intracellular domains. An alternative intracellular location of the carboxyl terminus (COOH) is designated should the 7th hydrophobic region not span the membrane (stippled area). Unit aa residues, (o), are notated in a-helix ( ~ ), ~sheet ( 88 ), or turn (ooo), with the corresponding charge (+/-) , if any. Predicted alternative secondary structures with equal propensities based on the Chou and Fasman (1978) algorithm are marked alongside the primary unit aa structure; ( " ), alternative a-helix; ( ), alternative [}-sheet, (. ,), alternative turn. (B) Helical wheel analysis of the seven putative membrane-spanning regions (Hj-H7) suggests a putative relatively hydrophilic pore. The number of hydrophilic residues lining the aqueous pore and those situated in the outer region are noted. The location of charged residues (+ or - ) is designated alongside the membrane-spanning regions.

is supported by the differences in codon usage and genomic DNA blot restriction fragment hybridization pattern. Chro- mosomal-mapping studies further support this conclusion. We have found that the three r subunit cDNA probes map to three different mouse chromosomes (Kent et al., 1987b). The high degree of nucleotide and aa sequence homology suggests that the three ~t subunit genes probably arose from a common ancestor. The sequence conservation of these three isoforms with other ATPases further supports the idea that different ion transport ATPases derived from a common ancestral gene (Serrano et al., 1986).

Significance of Na,K-ATPase a Subunit lsoform Diversity

The identification of isoform diversity exhibiting tissue- specific and developmental regulation is highly significant as it could provide the basis for Na,K-ATPase functional di- versity. This diversity includes markedly different tissue- specific responses to different physiologic conditions (Charle- magne et al., 1986) and hormonal regulation (Lytton et al., 1985), differences in ouabain affmity (Sweadner, 1985; Charle- magne et al., 1986), as well as specific cellular (Sweadner, 1979; Fambrough and Bayne, 1983) and subcellular localiza-

tion (Caplan et al., 1986). Direct support for this hypothesis awaits the determination of the functional characteristics as well as the cellular and subcellular location of each isoform. The differences in primary and secondary structure among isoforms, most notably in the amino termini and in proximity to the ATP-binding site may be involved in such isoform- specific functional characteristics.

Isoform diversity of the tt subunit raises the question as to the existence of isoform diversity for the I~ subunit. The pos- sibility of specific ~tx-I}x subunit quaternary associations could generate more complex assembly and functional inter- actions. Furthermore, the existence of r subunit isoform het- erogeneity described here makes it imperative to correlate enzymatic and pharmacologic parameters with the specific isoforms in order to assess respective physiological roles.

Structural Implications on the Mechanism of Ion Transport

Analysis of the topography of Na,K-ATPase a subunit, as de- termined by the identification of membrane-spanning regions, is central to the goal of eventually understanding the mecha- nism of ion transport. The membrane-spanning segments of the a subunit are most likely involved in the ion translocation

The Journal of Cell Biology, Volume 105, 1987 1862

process. However, the topology of Na,K-ATPase a subunit remains controversial. Kyte-Doolittle hydropathy plot analy- ses of three previously characterized a subunits (Shull et al., 1985; Kawakami et al., 1985; Ovchinnikov et al., 1986) have suggested different numbers of hydrophobic membrane- spanning regions. Because the Kyte-Doolittle scale does not address conformational and environmental aspects relevant to membrane proteins in the lipid bilayer (Engelman et al., 1986), we have reanalyzed all known a subunit aa sequences using the GES hydrophobicity scale. This scale has been pro- posed as appropriate for identifying nonpolar transbilayer helices in aa sequences of membrane proteins (Engelman et al., 1986). These analyses identified a consensus of seven putative membrane-spanning regions that can form a puta- tive hydrophilic pore. Although the accuracy of the different methods of relative amphiphilicity determination remain to be tested experimentally, the striking analogies between the transmembrane structures of Na,K-ATPase a subunit (pre- sented here) and bacteriorhodopsin suggest their validity. The latter has been shown by electron microscopy to have seven transmembrane helices (Henderson and Unwin, 1975), that are also identified by GES hydropathy analysis (Engel- man et al., 1986). Furthermore, neutron scattering data sug- gests the existence of a relatively hydrophilic pore (Engel- man and Zacai, 1980), that could provide a pathway for proton translocation (Tanford, 1982). More interestingly, the hydrophilicity of the putative pore defined by the Na,K- ATPase a subunit transmembrane segments is greater than the one in bacteriorhodopsin. Furthermore, hydrophobic regions H4, Hs, and H6, all greater than 20 aa (Fig. 9 B), are sufficiently long to cross the lipid bilayer in a path other than a perpendicular one. This may allow these transmem- brane segments to be involved in conformational changes im- portant in ion transport much like the tilting iris effect of the transmembrane helices of the gap junction in response to Ca ++ (Unwin and Ennis, 1984); or as proposed in the chem- ical potential change model of ion transport (Tanford, 1982). With the amino terminus previously shown to be in the cyto- plasmic side (Jorgensen et al., 1982a, b; Farley et al., 1986), the presence of seven hydrophobic regions places the car- boxyl terminus on the extracellular side. This putative to- pography more closely approximates the observation from previous biochemical data that the molecular mass of the ex- tracellular domain of Na,K-ATPase a subunit is •20-30% of the intracytoplasmic domain (Ovchinnikov et al., 1985).

Interestingly, Chou and Fasman (Chou and Fasman, 1978) predicted secondary structure propensity of the extramem- brane regions of al and a2 (Fig. 9 A; complete data not shown) shows that several regions have almost equal pre- dicted propensities to adopt an a-helix and/or 13-sheet and/or turn conformation. Changes between these two conforma- tions could be the structural basis for the E~-E2 conforma- tional transitions.

Na,K-ATPase a Subunit Interaction with Ouabain: Inhibition and Resistance

The binding of ouabain to the a subunit most likely involves two regions located between transmembrane regions H3 and H4 (1-OUA-R, Figs. 5 and 9 A), and between H5 and H6 (2-OUA-R, Figs. 5 and 9 A). These two regions are impli- cated by the location of a unique tryptophan (tryp 318, al) and cysteine residue (cys 809, al), respectively, previously shown to be involved in ouabain binding (Goeldner et al.,

1983; Kirley et al., 1986). There are no other extracellular tryp/cys residues between membrane-spanning regions. The predicted regions involved in ouabain binding are consistent with previous findings that the t~x~ E2 major tryptic pep- tides, amino terminus (41 kD) and carboxyl terminus (58 kD), are both covalently labeled with 3H-N-(ouabain)-N'- (2-nitro-4-azidophenyl) ethylenediamine (Jorgensen et al., 1982), and with the location proposed by 3H-anthroyl- ouabain fluorescence resonance energy transfer study (Cant- ley et al., 1982). The proximity of the putative regions in- volved in ouabain binding to transmembrane domains could result in steric hindrance of conformational changes involv- ing the respective flanking transmembrane regions by the glycoside. This hypothesis is consistent with the finding that ouabain binding keeps the Na,K-ATPase in the E2(K) form (Jorgensen, 1983).

Na,K-ATPases of rats, mice, and hamsters have been found to be ouabain resistant (Willis and Emory, 1983). Chromo- some-mediated gene transfer experiments suggest that only the al isoform segregates with the ouabain-resistant pheno- type. DNA sequences coding for the a2 and a3 isoforms and the 13 subunit were not transferred to recipient cells selected for ouabain resistance. Furthermore, direct transfer of mouse or rat al subunit cDNA is sufficient to confer ouabain resis- tance to ouabain-sensitive CV-1 cells (Kent et al., 1987a, b). These results suggest that the al isoform is responsible for the differential ouabain sensitivity among species (Fallows et al., 1987). Comparison of the rat al primary and secondary structures with the al equivalents from sheep and pig kidney, both ouabain-sensitive species, did not, however, reveal any insightful differences that could account for the differences in ouabain sensitivity. The two putative ouabain-binding regions and flanking aa sequences are highly homologous among the three species. On the other hand, there are differ- ences between the al type and the a2 isoform. The a2 iso- form, like the electric ray electroplax a subunit, has a non- charged residue (glycine) substituting for a charged one (glutamic acid) in the putative ouabain-binding region, 1-OUA-R (Fig. 5). Furthermore, in a2, an aspartic acid sub- stitution for valine in the extracellular-flanking region of the putative ouabain-binding region 2-OUA-R (Fig. 5) induces a six aa-long hydrophilic interruption in a hydrophobic stretch present in rat al, sheep, pig, and electric ray subunits. This hydrophilic region could affect ouabain binding.

In addition to al, ouabain resistance may also involve ex- pression of other a subunits isoform(s), as well as different levels of expression of these isoforms. Moreover, the ouabain resistance conferred by intrinsic differences in the a subunit isoforms must be distinguished from other phenomena ob- served in acquired ouabain resistance, such as gene am- plification (Emanuel et al., 1986; Pauw et al., 1986), and the expression of an ouabain resistance gene (Levenson et al., 1984).

The availability of cloned cDNAs for several a subunit iso- forms should allow the test of putative structure-function relationships and define the pharmacologic and biochemical properties of the a subunit isoforms through the expression of functional subunits from the isolated cDNAs and their in vitro-generated mutants.

We thank Dr. David Atkinson of the Biophysics Institute, Boston University Medical Center, for advice on the analysis of the protein structure, Ms. Lynna Stone for technical assistance on some of the work, Dr. Charles Sim-

Herrera et al. Na,K-ATPase (1 Subunit lsoforms 1863

mons for critical reading of the manuscript, and Ms. Sharon Ward for her excellent secretarial assistance.

This work was supported in part by grants (to B. Nadal-Ginard and R. Levinson) from the National Institutes of Health and the American Heart Association; by the Simeon Burt Wolbach Research Fund of the Children's Hospital of Boston (to V. L. Herrera). R. Levenson is an Established Inves- tigator of the American Heart Association.

Received for publication 17 December 1986, and in revised form 2 July 1987.

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