Sodium

cotransport

Ernest M. Wright, University

of California

proteins

Karl M. Hager and Eric Turk

at Los Angeles,

Los Angeles,

California,

USA

Significant advances have been made in elucidating the structure of Na+ cotransport proteins. Some fifteen of these low-abundance proteins have been cloned, sequenced and functionally expressed. They are members of the 12 membrane-spanning superfamily and they segregate into two groups, the Na+/glucose (SCLTI) and Na + /Cl-ICABA (CAT-I) families. SCLTI transporters are expressed in bacteria and animal cells, while CAT-1 transporters are mostly expressed in the brain. None have yet been found in plants.

Current

Opinion

in Cell

Biology

Introduction

fact

that

the cloning of the tirst euka~ote Na+ cotr;msporter [ 11, much progress has been made \\ith the cloning of other cotransporters. It is now e\-ident that there are at least wo unrelated gene families: one homologous to the intestinal and ren:d Na+ /glucose cotransporter ( SGLTl ), and another nith the brain Na+ ,/Cl-/GABA cotransporter (GAY1 1. The N:l+ cotrdnsporters belong to a clrlss of prok;t~0tic and eukar~.otic membrane proteins, the secondaT active transporters (cotransporters and antiporters ). The cotransporters (siiporters 1 iire responsible for the active accumulation of nutrients. neUrotT;inslllitters. vitamins, Me salts and ions within cells. The energ). to dri1.e the intracellular accumulation of these solutes is derived from the ion gradients (AL,, or A&+,) across the cell membrane, which are produced and maintained 131, primaq active transporters (H + and Naf J K+ pumps 1. In this re\ic? we summarize the recent progress with the Na + cotransporter proteins.

and

in’ in either is ;1 second

k~11306. between not used

is glycos~lated

;it Asn?tH

lying

in the

this

contributes

approsimatel!.

15 000

to the

ooc~~s or brush horder canonical amino-linked WI the putative intracellular transmemt~nlne segments [ 3.1,

~wmlxmes. glycos~lation hydrophilic 6 and 7, but

There site ;II loop this is

There are se\~~l consc’nsus sites for l~hos~~ho~lntion of SGLTl [i] 1 hut there is no c\%lence yet th:lt the!. pIa!. ;I functic~nal role. The) include sites on putative c-ytoplasmic hydrophilic domains ( Ser303 :ind Ser-r 18) that ma!’ be recognized by CAMP-, cGMl’and C- protein kineses. There are :llso two potenti: sites on external hydrophilic domains ( ThrSt and Seri66). hut these n~~ld he onI\. used if the seconda? structure moclel is incorrect or ;f there arc esternul kinascs. SGLTl has been functionall!~ expressed in .YvIIo~x~.< ooc~tes [ 1,~8,9**.10**], COS-7 cells [ 1 I 1, HeIa cells [ 111 and insect Sf9 cells [ 131. The substrate and ion specilicig’ and kinetics app’crlr to he quite independent of the expression system. This ~~)uld rule out impor tant effects of l”)st-t~lnslational moditicXions rind plasm:1 membrane composition on the t[‘:iiisporter function. The most comprehensive kinetic :lnalysis of SGLTl ws c;uried out hy Parent et trl. [9**.10**] using electrophysiological techniques. A six-stnte ordered kinetic model with mirror synimct~’ can cjuslitati\~el!. and clu:intitati\~el~ 3c‘count for Na+ glucose cotl’;insport: this has heen hased on measurements of prestead!. st:itc currents. sugar~specilic current-voltage (I V) cunw the voltage and Na+ dependence of sugar kinetics, the voltage ;ind sugar dependence of sodium kinetics. ;~nd the Na + leak currents

The the The

tirst m%unmali:tn cotransporters to he cloned lverc Klbhit and human Na+ glucose cotransporters [ 1 .L], amino acid sequence and :l seconda? structure model for the human SGLTl is shonn in Fig. I. The cDNA encodes a 664 residue protein that is predicted to cross the plasma membrane 12 times. Hence, SGLTl is a member of a large farnil!, of memhrune transport proteins. The sequence of SGLTI in m:unmals is highl>~ consen~ecl in both the small intestine and kiclne!- (Table 1 ). The amino terminus does not contain ;I reztdily identitiahle signal sequence [ 3.1, suggesting that it resides on the c-j.toplasmic face of the membrane. This is supported h!r the

Abbreviations GABA-y-amino butyrlc Biology

protein

mass of the mature ~iiemlxine protein 1-t 1. Perh:ips surprisingly, gl~cosylation is not required for transport acti\,

SCLTl

Current

the

Asn2+8,

family

@

4:696-702

hydrophilic domain hemeen transmembrrune segments 5 and 6 [3-j, I~iochcmical experiments indicate that major post-translational processing produces N-linked gl!,c~s)~lation of either the tri or tettxantennary comples gpe ;it

Since

The SCLTl

1992,

Ltd

ISSN

acid. 0955-0674

Sodium

Table

1. The

Na +/glucose

Na+-cotransport

family:

amino

acid

sequence

comparisons

of proteins

cotransport

proteins

homologous

to the

rabbit

Wright,

Amino Source

Na + /glucose

SCLTl

Identity

Rabbit

intestinal

(O/O)

Similarity

(%I

acids References

encoded

intestine

100

100

662

III

Kidney

100

100

662

115.161

Intestine

85

94

664

121

cells

84

92

662

1171

Rabbit Human LLC-PK, Na + /nucleoside

SNSTl

Rabbit

kidney

61

80

672

[19-l

Na + imyoinositol

SMITI

Rabbit

kidney

49

70

718

t20.1

Na + iproline

Put P

Escherkhia

26

56

502

I211

Na + ipantothenate

Pan F

22

53

402

[221

sequence

comparisons

co/i

E. co/i

are

relative

to the

rabbit

intestinal

sequence.

Sequence

alignments

were

obtained

as described

Fig. 1. A secondary structure model oi the human SCLTl The 661 residue protean is hhown with 12 transmembrane residues each. The protein IS glycosylated at Asn2JB and both the amino and carboxyl termlnt are shown facing the that detailed hydropathy analysis yields 11 13 transmembrane segments clepending on the algorithm used. absence of sugar. At 0 III\’ anti saturating and sugar, the rate-limiting step is the return of the empty carrier from the q-tosol to the external face of the niemhrane (5 scc ). but as the membrane is hyxxpolarized. the rate limiting step shifts to the dissociation of Na+ from the carrier at the c>-tosok face of the membrane. The most novel result was the recording of large transient currents ( tl ? z 5 ms ) when the membrane potential was rapidly depolarized in the absence of sugar (see Fig. 1. [W] ). These transients are probably due to the rapid reorientzition of the cotransponcr in the membrane electrical field, and they arc likely to be a general propew of cotransporters.

ohsen~~l csternal

Turk

cotransporter.

Transporter

The

Hager,

in the

N;I+

There is

in 121.

r-helices cytoplasm.

of 21 Note

an autosomal recessive disease of SGLTl called galactose malabsorption.Tt~e disease is characterized b!. a neonatal onset of severe diarrhea that is fatal unless glucose, galactose and lactose are removed from thv diet. The diarrhea results from a defect in the intestinal brush border Na+ ,‘glucose cotransporter. In the case of two sisters diagnosed with the disease, the trans. port defect was due to a mis-sense mutation that causes a change in residue 28 from aspartate to asparagine [I+*]. Note that an aspartate is conserved at the boundary beWeen the amino terminus and the lirst trdnsmembrane segment in a large number of transporters (see below). This is the first reported disease that is due to a mutation

glucose

697

698

Membrane

permeability

in a membrane transport protein. It is anticipated that analysis of other patients with glucoseigalactose malabsorption will provide more information about other essential residues for transport activiv.

SGLTl

family

members

Members of this family that have been identilied are listed in Table 1. Clones encoding proteins that are virtualI!. identical to SGLTl have been isolated from rabbit and pig kidney [ 15-171. These proteins are 662461 residues long and show at least 84% identity with SGLT-1. This similarity increases to more than 92% when consenative substitutions are taken into account. The rabbit intestinal and renal proteins are identical. It is unlikely that SGLTl is the major renal Na+,‘glucose cotmnsporter. Pajor et al. [ 181 examined the distribution of the rabbit renal cotransporter using brush border membmne vesicles, expression of kidney glucose transporters in oocytes. Northern blots and Western blots using mu polyclonal anti-peptide antibodies. SGLTl was found in the outer medulla, while another distinct glucose cotransporter was found in the outer cortex. On the basis of the Northern and Western blots it was estimated that the two renal transporters differed in sequence by more than 20%. Two other mammalian clones, the rabbit renal Na + nucleoside (SNSTI ) and Na+/myoinositol (SMIT) co transporters, show high homology to SGLTl, with 49 and 61% identity and 70 and 80% similarin’, respectively [ 19*,20*]. Neither of these transport cl-methyl-Dglucopyranoside, while SGLTl does not transport either myo-inositol or uridine. Surprisingly, phlorizin inhibits the transport actkity of both SGLTl and SMIT with inhibitor constants in the range of 5-100 ~mol/‘l. Hydropathy analysis indicates that SNSTl and SMIT belong to the 12 membrane-spanning family and that their secondaq structure profiles closely resemble SGLTI. In general, the secondary structure profile is quite distinct from other members of the 12 membrane-spannirlg superfam ily. For example, in the GLUT series (see Grifith et Al., this issue, pp 684695) there are two large hydrophilic loops between membrane spans l-2 and 6-7,while in the GAT-1 series the largest hydrophilic loop occurs between spans 3-i (see below). It also occurs in the Na+/Cl./GABA family where the largest hydrophilic loop is between spans 3-4 (see below). The SGLTl famil) is missing a hydrophilic carboxyl terminus. The most obvious difference between SMIT (718 residues) and SGLTl and SNSTl (662472 residues) is the length of the hydrophilic loops between membrane segments 6-7 and 11-12. The residues consenTed amongst the three cotransporters are shown in Fig. 2. The highest region of homology is in the first 550 residues, especially in membrane spans 2 and 8, and hydrophilic loops between membrane spans l-2, 2-3 and 7-8. Less than half of these conserved residues (101/255) reside in membrane domains, and, while most are hydrophobic, there are four amide, one acidic and two basic residues. These are obvious targets for site-directed mutagenesis experiments. The biggest difference between the proteins is towards

the carboq4 termini, especially in the large h!rdrophilic loop between spans 11-12 and in span 12. Note that Asp28 is conserved in all the mammalian members of this family. The similarities may point toward domains important in Na + and substrate binding and coupling. Finally, it should be noted that the putative phosphor).lation sites lie in hydrophilic loops between membrane spans G7 rind 8-9. Two bacterial Na+ cotransporters also belong to this family. Na + proline (Put I’) [ 2 1 ] and Na + :pantothenate (Pan F) 1221, although the regions of sequence homolocp;)are much lower (22-26’~ 1. In general, there is a substantial similariv between the kinetics of the mammalian and bacterial cotmnsporters [9**,10**,23]. The greatest kinetic difference lies in their alEnin for Na+ , (30 pmol/:l in bacteria and 30 mmol/ l in mammals). Between SGLTl , Put P and Pan F. 51 residues align, and although most of them are hydrophobic, three arginines, two serines, two tyrosines, one threonine and one aspartate cluster in three non-polar regions (6143,127-140,393+10~. In the 12 membrane model, membrane span 8 exhibits the greatest similariv bemeen the bacterial and mammalian cotransporters. Three other Na+ cotransporters halve been cloned: the bacterial Na+ , glutamate [2-t], rat li\.er Na+ ‘taurocholatc [ 35.1 and Na+ /‘phosphate cotransporters [ 26.1. There is no signiticant homoloQq between these and SGLTl, but at the 362 residue in the bile salt transporter a small region of homology with SGLTl and Put P has been noted ( 17-20% identity and 51-53% similariv). The bile salt and PO, transporters are anomalous, in that hydropathy anal>rsissuggest 6-8 membrane-sp:unning segments. Perhaps multimeric forms, e.g. homoclimers, are required for functional expression of these proteins. There is one report that SGLTl functions as a homotetramer [ 271. Despite the lack of overall sequence homolo~ between the glutamate, taurocholate and phosphate cotransporters, one structural motif, the SOB motif (Gly Ala X X X X Leu X X X GI) Arg, where X is any amino acid) is consemed in all but the bile acid transporter [ 241, The functional significance of this motif is not yet established, but Deguchi rt N/. [2-r] suggest that it is related to the sodium-binding site,

The CAT-1

family

GABA transporter

The second mammalian Na+ cotransporter to be cloned was the rat brain Na+ /Cl-/GABA cotransporter (GAT1 J. Guastella et &. [28] isolated the clone using an oligonucleotide probe based on the sequence of a peptide fragment from the purified transporter protein. The clone was expressed in oocytes and it induced Na+- and Cl- -dependent GABA transport with an afinity of 7 pmolil. The sequence predicted a protein of 599 residues, and hydropathy analysis indicated 11-13 transmembrane spanning domains. The secondary structure profile (Fig. 3) was quite distinct from that of other transporters, and there was no sequence homology with any

Sodium

Fig. 2. A 12 membrane-spanning In the models

membrane

domains

101

model residues

oi

rabbit SCLTl. are identical.

0, the 255 resldues Note that secondary

other protein in the database at that time. There has not yet been a detailed study of the kinetics and structure of the cloned transporter.

CAT-1

family

members

Seven related cotransporters (Table 2 1 1iai.e been cloned, sequenced ancl expressed in either ooqtes or cultured cells. The approach was either to use expression cloning [l] or to use polymerase chain reaction with oligonucleotides derived from the GABA transporter. The related proteins are mostly high afiniv neurotransmitter co-transporters (:itinity cx)nst:int: 0.3-100 pniol/l) from the central nervous system (biogenic amines, inhibitor? amino acids ). There is often an absolute requirement for chloride ions to carry out transport, which is why this related set of proteins is known collectively as the Na+ /Clcotransporter family. Previous work with synaptosonies and lmh slices has suggested that intracellular K+ is required for transport, but this has not yet been examined with the cloned proteins. One clone not isolated from the brain is the renal Na+ ,CI-:Betaine (GABA) cotransporter [ 29*]. This is involved in cell volume regulation in hypertonic media, and Northern blots show that this cotransporter is not expressed in brain. a tissue not exposed to variations in extracellular osmohity. All seven members of the GABA transporter family are 599-653 residue proteins with similar predicted secondary structure profiles, 1 I-13 transmembrane spans with a large extrxellular hydrophilic loop between spans 3-i (Fig. 3). This loop contains l-4 putative N-linked glycosylation sites. The amino acid sequences are all quite similar to GAT-1 (41-52 % identity and 66-73 % similar-

conserved structure

cotransport

between analyses

proteins

Wright,

Hager,

Turk

rabbit SCLTl, SNSTl and SMIT proteins. of SCLTI, SNSTI and SMIT yield similar

in,). There is higher homology between the dopamine [30*,31], serotonin [3P, 331 and noradrenaline transporters [3-r*] (4947 % identity). There is no detectable homology between the GAT-1 family, the SGLT-1 family or any other protein in the databases. This is especially surprising for the renal transporters involved in cell volume regulation in hypertonic media: the myoinositol (WIT) and betaine (RGT-1 ) cotransporters share no honiolofl~. As with the SGLT-1 faniil!Y, the homology within the GAT1 family is particularly noticeable in the amino-terminal half of the proteins. Transmembrane spans 1, 2, 5 and 6 are well consered ( ~13% amino acid identit),), mrhile spans 9-l 2 are poorly conserved, as is the glycosylated, extracellular, hydrophilic loop between membrane spans 3-4. In the GABA, dopamine, serotonin and noradrenaline transporters, 148 residues, many of which are situated in or close to the membrane, show sequence homo@gy. One glutamate residue is conserved in membrane span 10 in the four neurotransmitter transporters mentioned above, as well as in the renal betaine (GABA) transporter. These observations suggest that the glutamate residue may be essential for the operxion of the five carriers, and that the substrate specificity of the cotransporters is determined by the differences in the carboxy-terminal regions. Doubts have been raised, however, by the elegant studies of Mabjeesh and Kanner [35**], which show that the amino and carboxyl termini of the GABA transporter, possibly the transmembrane spans 1, 2 and 12, are not required for transport. They treated the partially purified protein with papain, removed the peptides from both terminals, which were identified by antipeptide amibodies, and reconstituted the core protein in liposomes. The activity of the core

699

700

Membrane

Table Na+

permeability

2. The ‘Cl

Na + Cl -

CABA

cotransport

family:

amino

acid

\equenc

e c ompancon~

ol proteins

homolr~gou~

to

Na + ICI - rc)kin. The advantage of this biochemical approach is that it bypassed the problems encuuntered 1,).expression of genetically engineered proteins, e.g. translation, processing, plasma membrane insertion and stabilit)~. Although there is no similariq~ between the SGLTl and GAT-1 cotransporters at the primaq~ or secondary setrrulsporter

rat

hasophiltc

leukemia Na+/noradrenallne

3’ sequences

the

cotransporter.

clwnce le\&. the ;tsIxtrf;tte residue ;it the c?-toplasmk interface bctmwn the xniino terminus and the tirst nwm brane span, Asp% in SGLI‘l, is conscn~ccl in 10 out of 15 Na+ cotrxqxxwrs. As 3 mut;ltic)n of A.y-13 t() AsnZX caused dcfcctkv sugar tT;tnyx)rt 1It**j. WC prv diet that Asp28 is essential for xtiviy in all transporters (not present in PO.,, Put I’, Pan F or glts ). I~reliniinq~

Sodium

esperinwnts demonstrxe that when Asp28 replied by A.sn28, Glu28 or Aia28. xctivity points to a structural role for Asp2X.

in SGLTl is is lost. This

This paper :I mcmhrane

clrscrilws protein.

Wright,

proteins

Hager, Turk

a methtxl to identi~ N-linked glycoq’lation sites in Only one of two potential sites is used in SGLTl.

-I

I IIRASAMA IsA. \Wc;irr Border Na + /glucose 1992. 11o!x3--l+.

i

K~:~sI~I~.Y I’l. KIWI{\ EG: Consensus Sequences 3s Substrate Specilicity Determinants for Protein Kinases and Protein Phosphates. ./ Hird C/XWI I99 I, 266: 1555% 1555X.

hs ma)’ be expected,

there is some owl-lap in the substrztte spcciticity of the GAT-1 trx-sporters. For example, srrrcosiw blocks proline upuke I-q, PROTl [ 36.1 and glycine upt:~lw by GLYTl [ 3?*] with inhibitor con st;ints in the range of l&50 ~niol,‘I, and cocxine inhibits the hiogenic amine transporters [ 30*.3 1,32-,343. There are cluantitati\v. I-. it not qciditati\v ditferences benVeen the trwsport charxteristics of the brain xd renal GAlSA trxq~ortcrs [ 7H,29*]: the dEnit\. constant for GABA up take \KI.S 7 ~molj I for GAT- I and 93 ~mol/ I for BGTl. ~cl nipecotic acid is ;I more potent inhibitor of GATI thw BGI‘I ( inhibitor constant XKI > 2000 ~mol I. resp’ectivel!,). It is clearl~~ neccssaq’ to es:lniine the suhstrnte specificit\. of these relatccl clones clc~sel!~. This is lxrticdad!- imlxxtxnt for the brain neiirotr:lnsmitters. where xXidepress3nts 31-e used to tre:it afftcti\v disorders. :tncl ilian~~ ahiisecl drugs. cg cocaine and amphet~umin~s. xc’ kno\\n to interxt \\ith the transporters.

cotransport

6

In~ir,\

TS. I I\\:\rc;

W’HIGIIT

porter I Io~x--9)5

Ehl. GIyco.sylation Cotransponer.

1: 5. Co:\~n

Ehl: Characterization Cloned from Rahhit

of the Hmchim

Rabbit Brush Hioplgfs AcIu

HA. I IEI~IGER MA. of a Nat /glucose CotransIntestine. ./ .I/wr/w Hi4 1989.

XIJ. ~IIIC\\‘A\LA

I’\II~u 11 jA. (:o.un \I!. W’w(;It’1 [