Thesodiumpumpbecomesa family Douglas M. Fambrough
The sodium pump, long recognized as central to the maintenance of Na + and K + ionic gradients, has gained a new personality, or we should say, set of personalities. Its catalytic subunit (o0 and probably also its glycoprotein subunit (f9 are encoded by several genes that are differentially expressed in different cell types. Of what physiological or regulatory relevance is this variety? The answers probably lie at many different levels of biological complexity. Cartoons depicting the (Na+,K+)-ATPase carrying out the obligatory counter-transport of Na + and K + across the plasma membrane now appear in every general biology textbook. The importance of this activity in maintaining osmotic balance and in setting the ionic gradients upon which the resting potential and action potential depend is widely appreciated. But far from being a relatively uninteresting 'housekeeping function', the sodium pump is certain to remain an important focus of research. There are a number of reasons, including the following. (1) The sodium pump is a relatively abundant plasma membrane protein in many cell types, with packing densities of up to at least 1000 molecules per wn 2 of surface membrane. This abundance facilitates all types of studies. (2) The sodium pump has a subunit structure complicated enough to attract investigators interested in membrane protein subunit assembly, yet simple enough to invite rigorous analysis. (3) As the prototypical cation transport ATPase, the sodium pump continues to be a challenge to those concerned with mechanisms of ion transport ~. (4) Because of its central role in the physiology of every cell, the sodium pump is subject to quite a variety of regulatory controls. Studies of these control mechanisms represent avenues into the analysis of basic cell processes such as regulation of membrane protein biosynthesis and degradation. (5) Regulation of the sodium pump2 involves not only setting levels of ion pumping capacity, but also establishing the spatial distributions of sodium pump molecules in the cell membrane. In polarized epithelia, the sodium pump generally is confined to the basolateral membrane. The distribution of sodium pumps in relation to the architecture of neurons, astrocytes, and myelinating glia remains hardly explored. Regulatory mechanisms involved in spatial organization are of prime importance in the nervous system, where cellular geometry is fundamental to function.
reticulum appear to have only a single, catalytic subunit homologous to the sodium pump a-subunit6. DNAs encoding a- and 13-subunits have recently been cloned and sequenced, providing new insights into sodium pump structure. The first reports of amino acid sequences for the e~subunits of sheep kidney7, pig kidney8 and Torpedo electric organ9 (Na+,K+)-ATPases, deduced from nucleotide sequences of encoding cDNAs, led to a new era in sodium pump studies. Since then, additional deduced sequences for humanz°, rat n-13, and chicken14 oc-subunits have been reported. Our laboratory has also determined sequences for the etsubunits of Drosophila (Lebovitz, R., unpublished observations). There has been an impressive conservation of primary sequence in the oc-subunit during evolution. Homology between Drosophila and the vertebrate oc-subunits is about 85% and among etsubunits of the various classes of vertebrates is 90% or better. Some regions show virtually no amino acid substitutions, particularly in the C-terminal half of the molecule. In extending our ideas of structure beyond the level of primary sequence to the level of secondary structure and membrane topology, the et-subunit provides a cautionary tale. Conjectures about the higher order structure of the (Na+,K+)-ATPase and homologous cation-transport ATPases involve models with the number of putative transmembrane segments ranging from six to twelve. The hydrophobicity plots from which these conjectures derive are virtually identical, not only for the various sodium pump oc-subunits but also for the approximately 100 kDa polypeptides of related 1 cation transport ATPases [the sarcoplasmic reticulum Ca2+-ATPases6, the gastric (K+,H+)-ATPase 15, and fungal plasma membrane H+-ATPases16'17], even though the primary sequences of these are rather divergent. There is less conservation of primary structure among the 13-subunits of the (Na+,K+)-ATPase from various vertebrate species (human18, dog19, pigs, rat 2°'21, sheep 22, Torpedo 2s, and chicken24). Overall sequence similarity between [S-subunits of different vertebrate classes is about 65%. However, the Nterminal domain (which is thought to be on the cytoplasmic side of the plasma membrane) and the single putative transmembrane domain are extremely well conserved throughout the vertebrates, as are specific aspects of the external domain, including the three N-glycosylation sites and the positions of the six cysteines.
Structure The (Na+,K+)-ATPase is generally thought to be comprised of o~- and [3-subunits3'4. However, the existence of a third type of subunit (y) has been suggested5, and there is lingering uncertainty about both the functional unit (0cd31or a2132)and the subunit stoichiometry. An added perplexity is the fact that no role in ion transport had been assigned to the 13subunit, and homologous cation-transport ATPases such as the Ca2+ ATPase of the sarcoplasmic
Multiple forms In 1979, Sweadner25 reported persuasive evidence that there were at least two molecular forms of the ctsubunit, differing in apparent molecular weight and in affinity for the cardiac glycoside strophanthidin. The slightly larger 'oc+' form, with greater affinity for strophanthidin, was the predominant form in the brain. In the past year it has become apparent that the a-subunit is actually encoded by a set of genes la,z6-2a thus accounting for the molecular heterogeneity of
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© 1988, Elsevier Publications, Cambridge0378-5912/88/$02.00
DouglasM. Fambroughis at the Departmentof Biology,Johns HopkinsUniversity, Baltimore,MD 21218, USA.
the oc-subunit in terms of differences in amino acid sequences among the isoforms encoded in separate genes. Rapid advances in this area have led to some problems in nomenclature, but now it appears that a homologous set of oc-subunit isoforms may exist in various vertebrate species. An oc-subunit nomenclature adapted from studies of rat o~-subunit isoforms by Shull et al. 11 is gaining widespread use: aI is the major kidney oc-isoform, odI (oc+ of Shull etal.) is a major aisoform in brain and skeletal muscle, and odII is expressed predominantly in the CNS. Our laboratory has carried out comparable studies on the oc-subunits of the chicken 14'29. The three avian oc-isoforms we have characterized most completely have amino acid sequence similarities to the three o~-subunit isoforms of the rat, including strong sequence similarities in the regions of maximum isoform-to-isoform variation, and there are even homologies in mRNA nucleotide sequences of 3' and 5' untranslated regions. Even the specificity of expression of oc-subunit isoforms in various tissues is similar in rat and chicken. These parallels suggest that multiplication of an ancestral ctsubunit gene occurred early in vertebrate evolution. The preservation of isoform-specific aspects of the ctsubunit amino acid sequence through evolution suggests that these different sequences may have important physiological relevance. Molecular heterogeneity of the ~-subunit presumably arises in part from differences in glycosylation between ~-subunits in different cell types 24. Additional molecular heterogeneity appears to be due to amino acid sequence differences: two N-terminal amino acid sequences have been found for the [3subunit in the chicken24. However, only a single type of encoding cDNA for a ~-subunit isoform has been cloned and sequenced from any one species. The occurrence of multiple genes encoding the ~-subunit is inferred fl-om the fact that monoclonal antibodies to the ~-subunit reveal different tissue distributions of ~subunits 29'3° (Fig. 1) and cDNA probes for the [3subunit detect little or no ~-mRNA in certain tissues (such as liver) where (in the case of the chicken) monoclonal antibodies reveal the presence of high levels of ~-subunit (Baron, R. and Fambrough, D., unpublished observations).
Fig. 1. Immunofluorescence micrographs showing immunologically distinct isoforms of oc- and fl-subunits expressed in skeletal muscle tissue. The panels show semi-serial cryosections. (A) and (B) show anti-at monoclonal antibodies; (C) and (D), anti-~ monoclonal antibodies. Abbreviations: N, peripheral nerve bundle; V, blood vessel;/H, skeletal muscle fibers. 326
How many genes encoding oc- and ~-subunits of the (Na +, K+)-ATPase are there? It is becoming commonplace to find a multigene family encoding a protein, and if we were to extrapolate from other instances, we would predict that the number of members of the 0~- and [3-suhunit multigene families would differ from one species to another. However, in different vertebrates, as mentioned above, there seem to be similar sets of ~-subunit genes. To account for the bulk of the ~- and [3-subunit protein in commonly studied vertebrate tissues, one needs to postulate a minimum of three oc-subunit genes (there may be more27'28), and a similar number of [3-subunit genes could be involved. The existence of multiple forms of each sodium pump subunit greatly complicates analyses of spatial distribution. To our knowledge no one has yet prepared a complete set of reagents with which to undertake a thoroughly rigorous description of isoform distribution. Nevertheless, some interesting observations are already in the literature. Immunological studies 31 point to different levels of expression of the od and 'oc+' (odI and/or odII3~) isoforms in the various neuronal cel types in rat retinal cells, whereas the Miller glia express only the od isoform. In skeletal muscle there is differential expression of immunologically distinct sodium pump isoforms in different muscle fiber types and in peripheral nerve 3° (Fig. 1). The regional distribution of encoding mRNAs in whole rat sections has been explored by in-situ hybridization with isoform-specific cDNA probes: among the major findings was localization of odII mRNA to neuronal cell bodies and embryonic cardiac muscle 32.
Regulation The existence of multiple isoforms of the sodium pump subunits also greatly complicates analysis of sodium pump regulation. Each cell type probably has its unique combination of regulatory controls on the sodium pump, including regulation of isoform selection, levels of gene expression, sensitivities to various modulators, and spatial distribution 2. In light of the molecular heterogeneity of the sodium pump, the task of describing its regulation in any cell must involve determining the array of subunit isoforms expressed by that cell type, together with a quantitative study of each isoform's behavior during a regulatory response. Obviously, isoform changes are an integral part of sodium pump regulation during development, since different adult cell types express one or another subset of isoforms 13'29-34. For example, in cardiac muscle a switch in expression of oc-isoforms seems to occur between fetal and postnatal life32'34. Might regulation of a sub-subset of these specific isoforms be involved in other cases of sodium pump regulation? In the case of insulin regulation of the sodium pump in adipocytes, Lytton35 has found evidence for two forms of oc-subunit, one of which has a Ko.5 for intracellular Na + transport of about 17 raM, whereas the other has a Ko.5 near 50 mM (and thus is essentially non-functional). Insulin stimulation appears to lower the Ko.5 of this second form, bringing it into play in ion transport. Differential regulation of subunits appears to be an important feature of up-regulation of the sodium pump in tissue-cultured chick skeletal muscle 29'3°'36. In this TINS, VoL 11, No. 7, 1988
cation transport system (measured as a6Rb+ uptake) with ouabain sensitivity identical to the avian type (IC5o about 2 ~M ouabain), operating in parallel to the mouse's ouabain-resistant system. When transfected with avian ~-subunit DNA, the mouse cells maintain their ouabain-resistant transport system (ICso about 200 ~tM). However, the avian ~-subunit, overexpressed in the mouse cells, appears to compete successfully with the mouse's own ~-subunit and form complexes with mouse ocsubunits so that the cells express a high level of interspecies hybrid avian-murine sodium pumps. The avian ~-subunit is properly Nglycosylated, the oligosaccharides are processed in a murine fashion, and the ct-[5 complexes are incorporated efficiently into the plasma membrane. These transfection results provide strong evidence that the ocsubunit is exclusively responsible for ouabain-sensitivity of ion transport, while the ~-subunit shows no obvious involvement. However, Fig. 2. Immunofluorescence microdetection of avian at-and flanother feature of the transfection scopical subunits expressed in mouse L cells results may lead to some enlighten- following transfections with their enment as to the role of the ~-subunit. coding cDNAs under control of the While virtually all of the avian i5- SV40 early promoter. Avian-specific subunit is expressed on the surface monoclonal antibodies to (A) aof the mouse cells, much of the subunits and (B) fl-subunits were avian et-subunit accumulates in used. Single cells, each expressing the endoplasmic reticulum (ER) only one of the avian subunits, are (Fig. 2). Perhaps, as we hypothe- shown. Note surface localization of sized above in the case of skeletal avian fl-subunits (B) compared with largely intraceflular localization of myotubes, the production of avian ac-subunits (,4). sodium pumps is rate-limited by iSsubunit biosynthesis. Over-production of a-subunits, then, would result in accumulation of unassembled aRoles of ~- and ~-subunits in ion transport and subunits. The appearance of high concentrations of bioregulation The multiple molecular forms of the sodium pump these in the ER is consistent with ~-subunits having a make analysis of ion transport difficult. To investigate role in the facilitation of intracellular transport of the function of individual isoforms and the contribu- oc-subulnts to the plasma membrane. Recent results by tions of each subunit to ion transport, three related Noguchi et al. 38 from expression of sodium pump strategies involving molecular genetic approaches are mRNAs in Xenopus oocytes are also consistent with being taken: (1) expression of sodium pump genes in the ~-subunit having this role. If the role of the ~-subunit is in intracellular organisms that lack (Na+,K÷)-ATPase, such as yeast; (2) expression of genes encoding ouabain- transport, then there are some interesting corollaries. resistant forms of the sodium pump in cells of a First, the Ca2+-ATPases of sarcoplasmic reticulum ouabain-sensitive organism; and (3) expression of (SR) and ER, although homologous to the (Na+,K+) genes encoding ouabain-sensitive forms of the ATPase, may remain in the ER and SR owing to a lack (Na+,K+)-ATPase in cells of a ouabain-resistant of affinity for any glycoprotein with ~-subunit function. organism. To date, the first strategy has yielded no Second, the cation transport ATPases homologous to results. Using the second strategy, Kent et al. 37 the (Na+,K+)-ATPase o~-subunit and present in the recently demonstrated that 9uabain-sensitive monkey plasma membrane might have some 'carrier' glycoprocells could be rendered ouabain-resistant by transfec- tein suhunit involved in transport to the cell surface. tion with DNA encoding a mouse oc-subunit. In In fact, it is even conceivable that the sodium pump 13implementing the third strategy, transfection of subunit fulfils this role for other cation transporters. Whatever the truth is, it is clear that the newly ouabain-resistant mouse L cells has been done with DNAs encoding o~-14 or [~-subunits24 of ouabain- appreciated molecular heterogeneity of the sodium sensitive avian sodium pumps. When transfected with pump must be taken into account in analysis of cation avian c~-subunit DNA only, the mouse cells develop a management in the nervous system. Fortunately, the
system, up-regulation can be triggered by verataidine, which opens the voltage-sensitive Na + channels and thereby allows continuous leakage of Na + into the fibers. The myotubes respond by increasing the number of sodium pumps in the sarcolemma, the number nearly doubling in 24 h. The signal perceived by the myotubes appears to be intracellular Na+; the mechanisms of transduction of this signal into the response remains largely a mystery. However, we now know that the response involves a selective increase in transcription of just the ~-subunit gene and a correlated increase in biosynthesis of the ~-subunit. Much of the resulting ~-subunit apparently never assembles with et-subunit. Although unassembled ~subunit is co-translafionaUy glycosylated at the three sites for N-glycosylation, these unassembled subunits do not continue through the biosynthetic pathway to the Golgi apparatus, but remain as high-mannose intermediate forms and are rapidly degraded. These phenomena suggest that the ~-subunit might be ratelimiting for assembly of new sodium pump molecules in the basal condition, and that during up-regulation, I~-subunit is over-produced and the ot-subunit becomes rate-limiting. Down-regulation in the skeletal muscle cultures can be effected simply by closing the voltage-sensitive Na ÷ channels with tetrodotoxin. Down-regulation rapidly returns the myotubes to the basal level of sodium pump expression (tl/2 = 3 h), and the mechanism for this change is interiorization of the 'excess' sodium pumps. If, as we imagine, the cells maintain excess ion transport capacity, then how does a myotube sense an abatement in demand for transport, and how does it manage to remove just enough sodium pump molecules from the sarcolemma to return the myotube to the basal level? One hypothesis is that the extra sodium pumps inserted into the plasma membrane during up-regulation are in some way a distinct subset and that the molecules are withdrawn from the surface when the demand for ion transport has abated. Could this hypothetical distinction be due to isoform differences?
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same molecular approaches that have revealed the extent of the complexity can be used to resolve the accompanying issues of relevance to regulatory mechanisms and physiology.
Selected references 1 Pedersen, P. L. and Carafoli, E. (1987) Trends Biochem. Sci.12, 146-150 2 Rossier, B. C., Geering, K. and Kraehenbuhl, J. P. (1987) Trends Biochem. Sci. 12,483-487 3 Glynn, I. M. (1985) in The Enzymes of Biological Membranes (Vol. 3) (Martonosi, A., ed.), pp. 35-114, Plenum Press 40vchinnikov, Y. A. (1987) Trends Biochem. Sci. 12,434-438 5 Collins, J. H. et al. (1982) Biochim. Biophys. Acta 686, 7-12 6 Brandl, C. J., Green, N. M., Korzak, 13.and MacLennan, D. H. (1986) Cell 44, 597-607 7 Shull, G. E., Schwartz, A. and Lingrel, J. B. (1985) Nature 316, 691-695 80vchinnikov, Y. A. et al. (1986) FEBS Lett. 201,237-245 9 Kawakami, K. et al. (1985) Nature 316, 733-736 10 Kawakami, K., Ohta, T., Nojima, H. and Nagano, K. (1986) J. Biochem. 100, 389-397 11 Shull, G. E., Greeb, J. and Lingrel, J. B. (1986) Biochemistry 26, 8124-8132 12 Hara, Y. etal. (1987)J. Biochem. 102, 43-58 13 Herrera, V. L. M., Emanuel, J. R., Ruiz-Opazo, N., Levenson, R. and NadaI-Ginard, B. (1987) J. Cell Biol. 105, 1855-1866 14 Takeyasu, K., Tamkun, M. M., Renaud, K. and Fambrough, D. M. (1988) J. Biol. Chem. 263, 4347-4354 15 Shull, G. E. and Lingrei, J. B. (1986) J. Biol. Chem. 261, 16788-16791 16 Addison, R. (1986)J. Biol. Chem. 262, 14896-14901 17 Serrano, R., Kielland-Brandt, M. C. and Fink, G. R. (1986) Nature 316, 691-695
18 Kawakami. K., Nojima, H., Ohta, T. and Nagano, K. (1986) Nucleic Acids Res. 14, 2833-2844 19 Brown, T. A., Horowitz, 13, Miller, R. P., McDonough, A. A. and Farley, R. A. (1987) Biochim. Biophys. Acta 912, 244253 20 Mercer, R. W. et al. (1986) MoL Cell Biol. 6, 3884-3890 21 Young, R. M., Shull, G. E. and Lingrel, J. B. (1987) J. Biol. Chem. 262, 4905-4910 22 Shull, G. E., Lane, L. K. and Lingrel, J. 13. (1986) Nature 321, 429-431 23 Noguchi, S. etal. (1986) FEBS Lett. 196, 315-320 24 Takeyasu, K., Tamkun, M. M., Siegel, N. R. and Fambrough, D. M. (1987) J. Biol. Chem. 262, 10733-10740 25 Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067 26 Kent, R. 13. etal. (1987) Proc. NatlAcad. 5ci. USA 84, 53695373 27 Shult, M. M. and Lingrel, J. B. (1987) Proc. Natl Acad. 5ci. USA 84, 4039-4043 28 Sverdlov, E. D. et al. (1987) FEB5 Lett. 217, 275-278 29 Takeyasu, K. et aL Curr. Top. A4embr. Transp. (in press) 30 Fambrough, D. M., Wolitzky, B. A., Tamkun, M. M. and Takeyasu, K. (1987) Kidney Int. 32, $97-$112 31 McGrail, K. M. and Sweadner, K. J. (1986) J. Neurosci. 6, 1272-1283 32 Schneider, J. W. etal. (1988) Proc. NatlAcad. 5ci. USA 85, 284-288 33 Young, R. M. and Lingrel, J. B. (1987) Biochem. Biophys. Res. Commun. 145, 52-58 34 Sweadner, K. J. and Farshi, S. K. (1987) Proc. NatlAcad. 5ci. USA 84, 8404-8407 35 Lytton, J. (1985) J. BioL Chem. 260, 10075-10080 36 Wolitzky, B. A. and Fambrough, D. M. (1986) J. Biol. Chem. 261, 9990-9999 37 Kent, R. B., Emanuel, J. R., Neriah, Y. B., Levenson, R. and Housman, D. E. (1987) Science 237, 901-903 38 Noguchi, S., Mishina, M., Kawamura, M. and Numa, S. (1987) FEB5 Left. 225, 27-32
The modulationof humanreflexesduringfundionalmotor tasks Richard B. Stein and Charles Capaday RichardB. Stein is at the Departmentof Physiology, University of Alberta, Edmonton, Canada T6G2H7, and Charles Capadayis at the Centrede Recherche en Sciences Neurologiques, University of Montreal, Quebec, CanadaH3C 3J7
Spinal reflexes are often viewed as stereotyped motor patterns with limited scope for modification. This presumed invariance is contrasted to the greater adaptive possibilities found in longer latency reflexes or voluntary reactions. However, recent evidence suggests that even short-latency, largely monosynaptic reflexes show a high degree of modulation during simple human motor activities such as walking and standing, and that the pattern of modulation can be specifically altered for the different functional requirements of each activity.
raised over many years ~, are beginning to emerge from studies of natural human motor activities. One prominent view, proposed by Feldman and his colleagues (recently summarized in an opencommentary format2) is that the modification of reflexes is limited to a shift in the threshold of a curve relating joint angle and torque. This view was derived from experimental work on antagonistic muscle groups working about the elbow. For simplicity, the concepts are illustrated in Fig. 1 for a single muscle and its reflexes, as the relationship between muscle In turning the pages of this magazine, voluntary length as an input and force as an output. The shape of commands are sent from higher centres such as the this relationship is defined by its threshold (the length motor cortex to the o~-motoneurons controlling the at which a force output is first seen) and its slope. arms and fingers. Sensory feedback also comes from Note that the slope may be different near threshold the skin, muscles and joints along sensory pathways than at higher output levels, but in Feldman's view the to the brain and, by reflex, to the motoneurons. Do shape of the input-output curve is fixed. For this these various centres function largely independently reason, he and his colleagues refer to the curve as the or do they interact in complex ways? For example, 'invariant characteristic'. Voluntary commands can during voluntary movements do higher brain centres then merely shift the whole curve along the x-axis also modulate the properties of reflex pathways? Is (i.e. change its threshold). The slope of the line relating length to force in Fig. the modulation specific to and of functional importance for the task being performed? If so, what are the 1 has the dimensions of stiffness. A contracting neural mechanisms used to produce this modulation? muscle has an intrinsic stiffness that is supplemented Answers to these questions, some of which have been by reflex responses. However, muscles generate © 1988, ElsevierPublications,Cambridge 0378- 5912/88/$02.00
TINS, Vol. 11, No. 7, 1988