Molecular biology of ion motive proteins in comparative models
Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA
* Author for correspondence (e-mail: michele.wheatly{at}wright.edu)
Accepted 8 June 2004
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Summary |
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Key words: ion, transport, channel, pump, cotransporter, antiporter.
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Introduction |
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Channels
Channels are open pores that allow free passage of the ion, based on
appropriate charge; they are not permanently open but are gated, typically by
ligands causing an allosteric change in the molecules that result in opening.
Proteins in this category include the epithelial Na+ channel
(ENaC), the epithelial Ca2+ channel (ECaC), the Cl-
channel (ClC) and the cystic fibrosis transmembrane conductance regulator
(CFTR, an apical, c-AMP regulated, low conductance Cl-
channel).
Pumps
Proteins in this category include Na+/K+-ATPase (the
Na+ pump), H+/K+-ATPase, the vacuolar
H+-ATPase (V-ATPase) and a range of Ca2+-ATPases on both
plasma (PMCA) and sarcoplasmic/endoplasmic reticulum (SR/ER) membranes
(SERCA). The well-studied basolateral Na+/K+-ATPase
transports and maintains an unequal distribution of Na+ and
K+ (3 Na+ out and 2 K+ in per ATP molecule
hydrolyzed) across the plasma membrane of nearly all eukaryotic cells, serving
an essential role in maintenance of osmotic balance and membrane potential. It
is worth mentioning that this protein, arguably the best studied of all ion
motive proteins, was originally described in nerves of the shore crab
Carcinus (Skou,
1957). Since then its three subunits (
, catalytic; ß,
targeting;
, regulatory) have been characterized in a range of species.
The apical V-ATPase was initially discovered in insect vacuolar membranes;
subsequently its role was broadened phylogenetically and it is now recognized
as an important energizer of apical plasma membranes in the same way that the
Na+ pump energizes the basolateral membrane in transporting
epithelia (Wieczorek et al.,
1999
). V-ATPases are known to acidify/alkalinize EC spaces of
polarized epithelia. In terms of Ca2+ pumps, sequence analysis has
focused on the SERCA pump, initially because of its abundance in muscle.
Subsequently it has been shown that SERCA plays an important role in
regulation of intracellular (IC) Ca2+ (sequestration in SR/ER),
allowing vectorial bulk flow through epithelial cells without toxic effect.
The basolateral PMCA regulates cytosolic Ca2+ levels by
transporting it against its electrochemical gradient using energy from
hydrolysis of ATP. The H+/K+-ATPase transports
K+ inward and H+ outward, leading to IC alkalinization
and EC acidification.
Cotransporters
Cation-chloride cotransporters perform a variety of physiological functions
including ion and cell volume regulation
(Mount et al., 1998). The
electroneutral Na+/Cl- cotransporter (NCC) is associated
with Cl- absorption. The
Na+/K+/2Cl- cotransporter (NKCC) effects net
inward salt transport in response to cell shrinkage. The
K+/Cl- cotransporter (KCC) couples movement of
K+ and Cl-. It is normally a net efflux pathway using
the favorable K+ chemical gradient maintained by the Na+
pump to drive Cl- out of the cell. The
Na+/HCO3- cotransporter (NBC) effects
acid-base regulation.
Antiporters/exchangers
The antiporters reviewed for this article include the
Na+/H+ exchanger (NHE), the
Na+/Ca2+ exchanger (NCX), the K+-dependent
Na+/Ca2+ exchangers (NCKX), the
Cl-/HCO3- exchanger and the
Na+-dependent anion exchanger (NDAE). The NHE is a reversible
electroneutral antiporter that functions in pH homeostasis, cell volume
regulation and transepithelial Na+ transport. The NCX regulates IC
Ca2+ at or below 100 nmol l-1 using the transmembrane
Na+ gradient as an energy source. NCXs are divided into two
families (NCX and NCKX), both of which are reversible. They usually couple
high external Na+ (NCX, 1Ca2+ for 3Na+,
electrogenic) or high external Na+ and high internal K+
(NCKX, 1K+ + 1Ca2+ out for 4Na+ in) to
transport Ca2+ out of the cell. Anion exchangers include
Cl-/OH-/HCO3- (pendrin), and the
NDAE.
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Unifying characteristics of ion motive proteins: diversity, versatility and interdependence |
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Certain ion motive proteins are ostensibly more important in the cellular
hierarchy than others. For example, the Na+ pump establishes the
electrochemical gradients used by apical and basolateral mechanisms such as
NHE, NKCC and various ion channels. Likewise the apical V-ATPase, through
establishing an electrochemical H+ gradient, drives Na+
uptake through apical ENaC or NHE. Ion motive proteins often accomplish
physiological functions other than ion regulation. For example ENaC, in
addition to mediating apical Na+ uptake, may serve in
mechanotransduction (Goodman and Schwartz,
2003). In addition to mediating transepithelial Cl-
transport, Cl- channels are involved in cell volume regulation,
stabilization of membrane potential, endocytosis and charge compensation
necessary for acidification of IC endosomes. In addition to being intimately
linked to acid-base balance, V-ATPases can energize fluid secretion.
Most ion motive proteins have multiple isoforms, often targeted to different cellular domains and with different function. For example there are typically two NKCC isoforms in vertebrates, one located basolaterally that is involved in volume regulation and NaCl secretion, and a second located apically and involved in NaCl uptake. A second example would be vertebrate NHE, where one isoform (NHE1) is located on the basolateral membrane and serves in IC pH regulation and volume regulation, whereas the apically located isoforms (NHE2/3) are involved in Na+ reabsorption and proton secretion.
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Comparative physiology of ion regulation |
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Adaptation to environment
Comparative models have historically informed the field of ion regulation
because non-mammalian organisms occupy a diversity of ionic environments
ranging from the Dead Sea (salinities several-fold that of seawater, SW) to
inland freshwaters (FW) and, in so doing, encounter unusually large ionic
gradients. The gills of aquatic species (particularly fish and crustaceans)
have become popular models for ion exchange due to issues of experimental
accessibility and simplicity. Teleost and elasmobranch fish have received
particular attention primarily because they are the oldest group of
vertebrates (500 million years, MY). Comparisons between related aquatic
species that inhabit environmental extremes can yield important mechanistic
information (FW crayfish vs SW lobster; FW vs SW teleost
fish). Euryhaline species possessing the ability to move between different
ionic environments (the euryhaline killifish Fundulus heteroclitus or
the portunid crab Callinectes sapidus) have engendered significant
interest, particularly those that routinely migrate as part of their
catadromous life history (for example migratory vs non-migratory
eels).
Mimic biomedicine
Other non-mammalian models have emerged due to unique anatomical structures
or processes that could be easily extrapolated to a given human process.
Examples of this would be: the urinary bladder of the toad serving as a model
for the mammalian renal collecting duct; the shark rectal gland and the avian
salt secreting gland that both mimic mammalian salt secreting epithelia; and
the killifish skin and gill epithelium that both mimic the mammalian airway
epithelium.
Extreme responses/anatomy
Other models have gained popularity based on some extreme response or
anatomical feature. In our laboratory, the moulting cycle of the aquatic
crustacean has emerged as a model to study Ca2+ transporting
proteins and the genes that encode them. The postmoult stage of the FW
crayfish Procambarus clarkii has presented a unique temporal `light
switch' for up/downregulating the Ca2+ motive proteins involved in
transepithelial Ca2+ fluxes required for rapid cuticular
remineralization (Wheatly,
1997). Other laboratories have extended the model to terrestrial
isopods (Porcellio scaber; Zeigler, 2002) that, in an effort to
conserve Ca2+ in terrestrial environments, moult the posterior and
anterior cuticle sequentially, necessitating both temporal and spatial
regulation of Ca2+ transport. In other cases the model has been
built upon some unique cellular or tissue attribute. For example the
Xenopus laevis oocyte has emerged as a model system for the
heterologous expression of a range of ion channels and transporters, primarily
because the cell diameter (1 mm) isaccessible for micropipettes/electrodes.
The simplified anatomy of the teleost renal tubule enables in vivo
studies of intact tubules due to the direct access to the renal portal system.
Other comparative models have been structured around a high abundance of a
particular ion motive protein (for example, the Na+ pump and NKCC
in shark rectal gland), which have led to the discovery of orthologues in
humans.
Not surprisingly, many of the comparative species for which sequence data
are available are organisms that have emerged historically as models for ion
transport. In some cases the molecular sequence of the ion motive protein was
first cloned in a comparative model. An example would be the electroneutral
NCC, which was first cloned and sequenced in the urinary bladder of the winter
flounder (Gamba et al., 1993).
In other cases sequences have emerged almost serendipitously. For example,
SERCA was initially selected for study by fish researchers as a way to
differentiate muscle fiber types rather than for any intrinsic interest in
Ca2+ regulation (Tullis and
Block, 1996
). Researchers using Artemia as a model to
study regulation of gene expression during embryonic development also selected
SERCA because the gene was abundant in the only accessible tissue in such a
small organism (Escalante and Sastre,
1994
). Not surprisingly, many ion motive proteins were first
cloned in mammalian models (ECaC, PMCA, SERCA, NHE), largely reflecting the
size of the research community and the application to human health.
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Comparative genomics of ion regulation: utility of genetic model organisms |
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The soil nematode C. elegans genome (97 Mb genomic sequence,
encoding over 19 000 genes) was the first to be completely sequenced and
published (The C. elegans
Sequencing Consortium, 1998) as a collaboration between the Sanger
Center (Wellcome Trust, UK) and the Genome Sequencing Center at Washington
University School of Medicine, USA. In the 1960s Sydney Brenner recognized
that this primitive species could provide a useful model for the genetics of
development and neurobiology in humans. The worm is conceived as a single
cell, which undergoes embryonic cleavage, morphogenesis, development, nerve
function, behaviour and aging - in a nutshell encompassing all the mysteries
of modern biology. In the case of C. elegans, developmental studies
are simplified because the identity, position, lineage and fate of every
somatic cell (959 in total) are known through embryogenesis and larval
development. Not only are genetic and reverse genetic screens possible, but
techniques such as double-stranded RNA-mediated gene interference also allow
researchers to phenocopy a null allele in the space of days. This metazoan is
unique in that it can be grown and genetically manipulated with the speed and
ease of a microorganism, yet it offers many features common to higher
organisms.
The C. elegans sequence was followed within a year by the
completed genome of the fruitfly, Drosophila melanogaster, a species
that had been employed as a genetic model for almost a century. The D.
melanogaster genome was sequenced as a collaboration between Celera and
Berkeley Drosophila Genome Project and was first published in 2000
(Adams et al., 2000). As with
C. elegans, Drosophila has a genetic system that is easy to
manipulate, can be maintained at relatively low cost, and affords biological
complexity that is believed to parallel mammals.
By comparison, the Anopheles genome project attributes its origins
less to an existing community of genetics researchers and more to the role
this species plays as vector in the worldwide spread of malaria. The need to
control the spread of this disease and to develop improved antimalarial drugs
and vaccines has fuelled this project. The genome sequence was a collaboration
between several sequencing centers and was supported by the World Health
Organization (Holt et al.,
2002).
The sea squirt Ciona intestinales initially appears to be a most
unlikely candidate for genomics; however it transpires that it has the
smallest genome (estimated size 155 Mb) of any manipulable chordate and
provides the opportunity to explore the evolutionary origins of the chordates
(over 550 MY) through easily visualized cells and transient transgene
expression. A draft of the genome was published by Joint Genomics Institute,
US Department of Energy (Dehal et al.,
2002).
Likewise the Japanese puffer fish Fugu (or Takifugu)
rubripes has emerged as a model genetic species since it possesses
one of the smallest genomes of all vertebrates (400 Mb, or one eighth the size
of the human genome), attributable to a compactness of introns, intergenic
distances and marked reduction of repetitive DNA sequences. A preliminary
analysis of the genome is reported in Science headed by the Joint
Genomics Institute (Aparicio et al.,
2002). It was the first vertebrate genome available following the
sequencing of the human genome in 2001. Sequence comparison will enable
interpretation of the human genome, assist drug discovery and inform the
evolution of vertebrate genomes over the 450 MY since the species diverged
from a common ancestor.
A powerful combination of genetics and embryology has established the zebrafish Danio rerio as an important model organism for the analysis of vertebrate development, physiology and behavior. The transparency and external development of the embryo allow exquisite manipulations such as dye-labelling, transplantation and in vivo time-lapse imaging that illuminate the function of mutated genes at the cellular level.
The zebrafish sequencing project was commenced in 2001 at the Sanger Institute and the completed genome sequence is anticipated at the end of 2005. With a genome that is half the size of mammalian models (1.6-1.7 Gb), it promises to serve as a model for human biology and disease.
In addition to the large-scale genomic sequencing projects, EST (expressed sequence tags) and cDNA sequencing projects are becoming available for a growing number of additional species. Within the Arthropoda this would include several insect species that are associated with disease transmission [such as the mosquitos (A. aegypti, Dengue fever; A. albopictus, Dengue fever; A. triseriatus, encephalitis; Culex pipiens, West Nile virus) the tick, Amblyomma americanum and the tsetse fly Glossina morsitans, sleeping sickness] or have agricultural importance (such as the honey bee Apis mellifera, the silkworm Bombyx mori and the beetle Triboleum castanum). Additionally two frog species are being studied as models of vertebrate embryonic development (Xenopus laevis and X. tropicalis). Just like the large scale genomics projects, these also support emerging internet resources (Table 1).
As genomes of additional model species are sequenced (at the predicted future rate of several each year), around several thousand putative `transport' proteins (as much as one fourth of the genome) are deposited into the genomics databases at one time. While the genomics data are expanding exponentially, annotation is following at a snail's pace. Genes are often assigned to gene families on genome-specific databases where they await confirmation of gene identities for inclusion in the GenBank. Even so the available sequences offer great opportunities for homology-based cloning, especially for researchers who study either arthropods or chordates.
In most cases the nucleotide sequences of the ion motive proteins of
genetic species have resulted as by-products of the genome sequencing project
and less for any intrinsic value in furthering the understanding of ion
transport. A good example of this would be the early cloning of SERCA in
Drosophila that was rapidly followed by its gene mapping to the right
arm of chromosome 2 at band 60A-B (Magyar
and Varadi, 1990). Subsequently the gene was characterized based
on accessibility of genomic libraries
(Magyar et al., 1995
). On the
other hand, since these genome projects are spread widely through the animal
kingdom, it has facilitated the work on non-genetic species since it is now
possible to identify candidate cDNAs in the closest genetic organism. In some
cases sequences from genetic models have led to the mammalian equivalents. A
good example would be the NDAE, which was first discovered in
Drosophila (Romero et al.,
2000
) and may assist in the molecular identification of other
cation and anion-coupled HCO3- transporters. Its
discovery in Drosophila has enabled genetic manipulation, suggesting
that disruption is lethal.
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Role of genetic organisms in the post-genomics era: closing the phenotype gap |
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An excellent review by Dow and Davies
(2003) illustrates how the
extensive genomic resources of Drosophila have already informed the
structure, function and control of epithelial ion transport in the insect
Malpighian tubule, a tissue with functional similarities to the mammalian
kidney. For example, they describe how reverse genetics in Drosophila
illuminated the role of V-ATPase through a knock-out that enabled
characterization of expression patterns in tissues and cell types. Further,
they illustrate how enhancer trapping (ET) technology can be used to detect
tubule-specific enhancers in the genome that can provide insight into the
spatial organization of this tissue. Put simply, an ET element is a
transposable DNA element (usually a P element) that inserts at scattered sites
throughout the genome close to an endogenous enhancer of gene expression. For
enhancer trapping, a P element is used in which the transposase gene has been
replaced by three elements: a visible marker enabling new insertions to be
tracked through crossing schemes (white+, for example, confers red eye color
in a white-genetic background), a reporter gene (such as lacZ) whose
expression is readily visualized, and an E. coli origin of
replication to facilitate plasmid-rescue of the flanking sequences. Without a
source of transposase the element is trapped within the genome; this enzyme
can be provided by crossing flies carrying the P-element to another line in
which it has been inactivated. `Jump-starters' are flies (typically males)
that carry both the ET element and a source of transposase. By breeding true
from thousands of progeny of such a male, it is possible to look for
interesting patterns of expression of the reporter gene in the tissue of
interest. Those authors screened 1500 lines with the P{GATB} transposon and
discovered 20 lines that were subsequently used to identify genes of interest
in the Malpighian tubule (much akin to a differential cDNA library screen).
The technique has subsequently illuminated the regional specialization and
multiple cell types in this important epithelial tissue.
A second example from Drosophila would be the parallels between
the insect tracheal system and the mammalian airways. Both consist of
branching networks of tubular epithelia delivering oxygen to respiring cells,
which must convert during development from liquid- to air-filled systems
through the common mechanism of active salt absorption using ENaC. As such,
the genetic model Drosophila has served as an excellent non-mammalian
species to better understand the role of ENaC during the water-to-air
transition (Liu et al.,
2003).
Two examples can be used to illustrate how C. elegans research has
informed the physiology of ion motive proteins, specifically ClC and NHE. One
of the advantages of C. elegans as a model system is that both
genetic and reverse genetic screens are accessible. Researchers can rapidly
generate cell-specific antisense inhibition of a given ClC isoform or employ
RNAi knockdown of message levels along with in situ patch clamp of
the worm, enabling them to answer very specific questions about the role of
ClC such as expression patterns and properties of the individual isoforms
(Schriever et al., 1999;
Nehrke et al., 2000
). C.
elegans has also contributed to the identification and understanding of
NHE homologues (Nehrke and Melvin,
2002
), specifically whether certain isoforms are expressed at the
cell surface or on membranes of IC organelles and whether the particular
characteristics of an isoform may be tailored to the functions exclusive to a
single cell type.
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Role of non-genetic organisms in the post-genomics era: comparative, environmental and evolutionary genomics |
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Aligning differences in function with differences in sequence
In situations where the mammalian sequence is already known, cloning and
sequencing the same ion motive protein in an evolutionarily distant species
can provide valuable information, particularly concerning differences in
functional characteristics or sequence homology. A good case study to
illustrate this point would be the NCX (He
et al., 1998). NCX was first physiologically characterized in
squid giant axon. In due course the canine cardiac NCX was sequenced
(Nicoll et al., 1990
). What is
the value in now cloning the NCX from squid axon? While some of the basic
properties of the squid and canine cardiac NCX are conserved [stoichiometry,
Na+-dependent inactivation, secondary activation by cytoplasmic
Ca2+, and deregulation by chymotrypsin, stimulation by ATP and
phosphatidylinositol (4,5)-bisphosphate (PIP2)], there are
important differences between the two. In squid the ATP dependence involves
phosphorylation by protein kinase whereas in canine cardiac NCX it reflects
generation of PIP2 from phosphatidylinositol. Further ATP
S
activates the squid NCX but not canine cardiac NCX. Cationic agents that bind
anionic lipids inhibit the canine cardiac NCX but agents like pentalysine do
not inhibit the squid NCX. The squid NCX is regulated by a
phosphoarginine-dependent process that may involve protein kinases unique to
invertebrates whereas it has no effect on canine cardiac NCX. In squid the
Ca-Ca exchange operation of the NCX is voltage dependent while the Na-Na
exchange is not. In sum, there are interesting functional differences between
the NCX proteins of these two species.
When the squid NCX was sequenced it exhibited about 58% identity with
canine cardiac NCX and regions determined to be of functional importance were
well conserved [specific acidic residues within the binding site for
regulatory Ca2+, endogenous exchanger inhibitory peptide (XIP)
region involved in Na+-dependent inactivation, predicted topology].
Sequence conservation was highest in the proposed transmembrane segments 2, 3,
8, 9 (the repeats) consistent with a catalytic role of the hydrophobic
domains in ion translocation. However there was a noticeable deletion of 47
residues in the large IC loop. In mammalian NCX this region displays extensive
alternative splicing. Transmembrane region (TM) 11 is the least conserved
domain, suggesting that the C terminus plays a lesser role in exchanger
function. Also the N terminus, which represents a signal peptide region, is
poorly conserved among NCX proteins. The molecular basis of differences in the
voltage dependence of canine cardiac NCX and squid NCX can now be pursued by
the combined methods of molecular biology and electrophysiology.
Tracing the evolution of important gene families
Comparative genomics of species separated over evolutionary time can also
assist in reconstructing the history of gene families. An example from our own
work would be the evolution of SERCA pumps
(Wheatly et al., 2001). In
vertebrates there are three homologous alternatively spliced genes that encode
five isoforms. By comparison in invertebrates a single gene transcript has
been identified in C. elegans (Cho
et al., 2000
), Drosophila
(Varadi et al., 1989
;
Magyar et al., 1995
),
Artemia (Palmero and Sastre,
1989
; Escalante and Sastre,
1993
) and Procambarus
(Zhang et al., 2000
;
Chen et al., 2002
). In
nematode, brine shrimp and crayfish, two isoforms originate from this
transcript. In C. elegans both isoforms appear to be essential for
embryonic development and post-embryonic growth and survival. In brine shrimp
and crayfish the two isoforms are homologous to vertebrate SERCA2a and
SERCA2b, and originate by the same alternative splicing mechanism. Put simply,
the final six amino acids of one isoform are replaced in the other isoform by
an extended C terminus of 30 residues, possessing hydrophobic properties that
could potentially form an additional transmembrane domain. There is no
evidence to show that the carboxyl terminus of a Ca2+ pump is vital
to function and yet diverse species have conserved alternative SERCA2a and b
termini through millions of years, suggesting that there is some selective
advantage to retaining multiple isoforms. Before the ancestors of crustaceans
and vertebrates diverged 600 MY ago, they probably shared the same ancestral
SERCA gene with a charged C terminus that gave rise to three homologues
(SERCA1, 2, 3) in vertebrates; the same alternative splicing mechanism was
retained in the invertebrate gene and in the SERCA2 vertebrate gene while it
was lost in the other two homologues (SERCA1 and SERCA3). The vertebrate genes
for SERCA1 and 2 have unique promoters for transcription of the two isoforms
encoded by each gene. The generation of one or other isoform is dependent upon
the processing of the last exons of each gene that has been shown to be
tissue-specific for SERCA2. In Artemia the expression of the two
protein isoforms is regulated at the transcription initiation step
(Escalante and Sastre, 1995
).
There are two different promoters that independently regulate the expression
of each isoform. A second mechanism is the differential processing of the last
two exons of the gene, which is also tissue specific, and this mechanism has
obviously been conserved between Artemia and vertebrates.
The reverse is also true, namely that the analysis of ancient protein
families can aid our understanding of phylogeny. To continue with the case of
the SERCA pumps, an analysis by Hagedorn et al.
(2003) confirmed that the
sequences for the malacostracans Porcellio and Procambarus
are more closely related to insects than to the branchiopod crustacean
Artemia. The further study of other sequences from arthropods may
help to resolve the controversy about the monophyletic origin of Crustacea.
Some hypotheses have suggested that the Branchiopoda branched off the main
stem of Crustacea before the insects.
The phylogenetic analysis of ion motive proteins can be useful for
biomedicine. An example of this would be cystic fibrosis, which is a common
lethal autosomal recessive disease caused by mutations of the CFTR gene.
Sequence comparison can illumine the structure-function of the protein, which
will facilitate interpretation of the identified mutations in the gene. A
recent analysis by Chen et al.
(2001) of 16 full-length CFTR
amino acid sequences from a wide range of vertebrate species representing up
to 420 MY of evolution, has enabled a functional R domain to be defined
(phosphorylation of this domain regulates channel activity), redefined the
boundaries of the two nucleotide-binding domains and the C-terminal tail,
provided insights into the differential roles of the two halves of CFTR, and
identified several well-conserved motifs that may be involved in inter- or
intramolecular interactions. Such phylogenetic analysis will enable
appropriate animal models to be selected for further in-depth analysis of
complex CFTR defects, possibly leading to a better treatment of the
disease.
Environmental genomics: aligning molecular biology with environmental need
The application of the Krogh principle will persist into the postgenomics
era as researchers seek to learn how the molecular biology of an ion motive
protein has evolved to fit environmental need. As in the past, organisms that
are suited for life in hostile or unusual environments will inform the general
understanding of these important proteins.
An example of this would be the ability of rainbow trout Oncorhynchus
mykiss to maintain heart contractility under hypothermic conditions
(4-15°C) that would be cardioplegic to mammals. Differential temperature
dependencies in the mammalian vs teleost NCX are due to important
differences in the primary structure of the isoforms
(Xue et al., 1999). Knowledge
of the sequence and biophysical properties of trout heart NCX can contribute
to the understanding of the evolution of the NCX over 400 MY.
Comparisons would not necessarily be restricted to organisms separated so
widely in evolutionary history. Closely related organisms that have ion motive
proteins with different functional characteristics can be equally informative.
An example would be the comparison between SERCA sequences in a
freeze-tolerant frog, Rana sylvatica, vs a related cold-intolerant
species, Rana clamitans. Muscle contractility at low environmental
temperatures in R. sylvatica has been attributed both to functional
(exhibits double the activity at 0°C and a lower activation energy below
20°C) and structural differences in SERCA1
(Dode et al., 2001). Sequence
analysis revealed that R. sylvatica has two amino acids in SERCA1
that are unique, three of which are located in the ATP binding domain. These
differences may shed light on the temperature dependence of ATP hydrolysis,
kinetics with ATP, Ca2+ cooperativity and protein-protein
interactions.
Regions of sequence difference cannot always explain the functional
differences between molecules operating in different environments. For example
CFTR sequence analysis has benefited from a comparison of amphibians and
mammals (Price et al., 1996).
In amphibians, which are adapted to living in FW, CFTR is located in the
apical membrane of skin and urinary tract. Anion selectivity and
pharmacological profile are both different to that of human CFTR. The R domain
is the most divergent region between the two sequences, although
phosphorylation sites responsible for protein kinase (PKA)-dependent
regulation are conserved within the R domains. Much of the R domain can be
deleted without disrupting channel activity. In this case differences in
sequence obviously do not prevent correct folding and association between
different domains.
In other cases sequence differences may reflect a combination of
evolutionary history and/or environmental adaptation. For example the low
sequence homology of the ray Na+ pump subunit with those of
teleost fish, amphibians, birds and mammals may be due either to evolutionary
age of the elasmobranch class of fish or, alternatively, to structural
adaptation of these proteins to function in the presence of high circulating
urea concentration (Cutler et al.,
1995
).
Providing technical advantage in spatial and temporal expression analysis
To come full circle, just as comparative models have aided the early
understanding of ion regulation, they will continue to provide technical
advantages in understanding expression patterns of gene products, largely
because of experimental accessibility.
A good example would be the study of ClC, whose functional characteristics
are poorly understood. Chloride channels (ClC5) in both Xenopus and
human cells have similar functional properties. Amphibian renal A6 cells have
proved useful in studying trafficking and functional regulation of ClC-5. Mo
et al. (1999) compared ClC-5
in amphibian renal A6 cells with human cells using isogenic constructs
consisting of an open reading frame subcloned into an optimized
Xenopus expression vector. The Xenopus clone resulted in
strong rectifying outward currents that were not affected by Cl-
channel blockers. The anion conductivity sequence was
NO3->Cl->I->HCO3-.
A reduction in EC pH inhibited outward currents. Since ClC are often
colocalized with H+-ATPase in IC vesicles below the brush border,
it has been suggested that these channels facilitate the acidification of
endosomes (route for Cl- influx as a counterion to H+
transport).
Some unique expression property of a given tissue/cell type may dictate the
need for a comparative model system in gaining insight into functional
genomics. For example, the urothelium of Bufo can coexpress different
Na+ pump ß subunit isoforms
(Jaisser et al., 1992), only
one of which is regulated by aldosterone. This model can assist in dissecting
out the physiological relevance of different isoforms. A second example would
be the existence of two populations of intercalated cells in turtle bladder.
The
cells (acidifying) express apical V-ATPase that drives
H+ excretion and HCO3- reabsorption
via a Cl-/HCO3- exchanger; the
ß cells (alkalinizing) express basolateral V-ATPase involved in
HCO3- excretion and Cl- uptake via
an apical Cl-/HCO3- exchanger
(Stetson and Steinmetz,
1985
).
Expression of many ion motive proteins is being advanced through continued
examination of aquatic species inhabiting a range of ionic environments. The
simplicity of the gill can enable experimental approaches not afforded by
`higher' organisms. For example, a range of fishes with differing abilities to
ionoregulate (Myxine glutinosa, Raja erinacea, Fundulus heteroclitus)
are being used to further explore NHE expression trends
(Edwards et al., 2001;
Choe et al., 2002
). Similarly a
spectrum of crustaceans with different ionoregulatory abilities are being used
to better understand the regulation of expression of V-ATPase
(Weihrauch et al., 2001
) and
Na+/K+-ATPase (Towle
et al., 2001
).
NHE has been selected to illustrate how comparative species continue to
advance our understanding of physiological function, location of isoforms and
mechanisms of regulation. Claiborne et al.
(1999) has utilized a range of
teleost species to explore the competing demands placed on NHE for
Na+ transport and acid-base balance, depending upon the location of
the isoform (apical NHE2 vs basal NHE1). So, for example, metabolic
acidosis caused a decrease in expression of basal NHE1 in sculpin to enhance
net H+ transfer to the water via the apical NHE2 isoform.
While Na+ uptake across the gills may appear inappropriate when in
SW, it constitutes only a portion of total Na+ influx and may be
worth the additional energetic costs to maintain acid-base balance. A third
isoform, ß-NHE, is involved in IC housekeeping (pH homeostasis and volume
regulation). This isoform is activated by cAMP, which inhibits activity of
apical NHE isoforms and does not affect those on the basolateral membrane.
These differences in regulatory mechanisms can be explained either by the
existence of different isoforms or the fact that there is one form of the
exchanger and that differences in regulation are dictated by the cell type and
different signaling networks. Parallel studies in crustaceans have revealed an
NHE3-like isoform in gills of the crabs Carcinus and
Callinectes (Towle et al.,
1997
) that may equate to the electrogenic exchanger
(2Na+/1H+) documented in physiological studies. In the
case of crustaceans there is evidence for both electrogenic and electroneutral
NHE on both the apical and basolateral membranes.
Colocalization in comparative species can also shed light on the
interdependence of the ion motive proteins. For example the V-ATPase and ENaC
were colocalized on apical membranes of pavement and chloride cells in FW
tilapia Oreochromis mossambicus, suggesting that they may be linked
in function (Wilson et al.,
2000). In the same vein, separation of localization can restrict
function to different cell populations. In stingrays (Dasyatis
sabina; Piermarini and Evans,
2001
) V-ATPase abundance was greatest in FW-acclimated animals
associated with increased NaCl uptake and
H+/HCO3- extrusion. Localization occurred
diffusely throughout the cytoplasm and was associated with the basolateral
membrane of large mitochondria-rich cells. VATPase and
Na+/K+-ATPase were located in different populations of
cells, suggesting that Cl- uptake/HCO3-
excretion occurs in V-ATPase-rich cells while Na+
uptake/H+ excretion occur in Na+/K+-ATPase
rich cells. In a subsequent paper
(Piermarini et al., 2002
) the
same authors established that the Cl-/HCO3-
exchanger (pendrin) immunoreactivity occurred at the apical region of
V-ATPase-rich cells and not in the Na+ pump-rich cells.
Comparison of expression patterns in response to environmental change can
further our understanding of the regulation of the genes that encode these
proteins. The subunit of Na+/K+-ATPase in
Callinectes exhibits greater expression in posterior gills than in
anterior gills (Towle et al.,
2001
). When these euryhaline crabs are exposed to external
dilution, their ability to hyperosmoregulate is associated with enhanced
Na+ pump activity in posterior gills. However, the documented
increase in enzymatic activity is not reflected in increased expression of the
mRNA. This suggests that post-translational mechanisms are responsible for the
increased activity (such as subunit assembly, membrane trafficking or cell
signaling) and not synthesis of new protein.
Certain comparative models avail themselves to the analysis of temporal
regulation of expression of ion motive proteins. In our own laboratory the
crayfish moulting model has shown that Ca2+-ATPases on internal
(SERCA) and external (PMCA) membranes appear to be inversely regulated. SERCA
expression is greatest in intermoult and decreases in postmoult whereas PMCA
expression is lower in intermoult and increases in postmoult
(Zhang et al., 2000). This
agrees with the pattern observed in mammalian cells
(Liu et al., 1996
) where SERCA
expression was downregulated when PMCA was functionally overexpressed in rat
aortic endothelial cells. In the terrestrial isopod model, Porcellio
scaber (Ziegler et al.,
2002
), SERCA expression showed the opposing trend. Messenger RNA
abundance was increased during the change from non-transporting (early
premoult) to Ca2+ transporting stages of the moulting cycle (late
premoult to intramoult). Expression of SERCA in the anterior sternal
epithelium (ASE) was different from the posterior sternal epithelium (PSE),
suggesting that there is tissue specific regulation.
![]() |
Summary |
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|
![]() |
List of abbreviations |
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![]() |
Acknowledgments |
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This Commentary was commissioned by the late Bob Boutilier and is dedicated to his memory.
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