OPINION
Water channel proteins: from their discovery in Cluj-Napoca,
Romania in 1985, to the 2003 Nobel Prize in chemistry andtheir implications in molecular medicine
Gheorghe Benga
Department of Cell and Molecular Biology, ‘‘Iuliu Hat,ieganu’’ University of Medicine and Pharmacy
Cluj-Napoca, Cluj-Napoca, Romania(Received for publication on November 8, 2005)(Revised for publication on February 22, 2006)(Accepted for publication on February 22, 2006)

Key words: membranes, water channels, water channel proteins, aquaporins, red blood cell

membranes. In the ‘‘Early Period of Membraneology’’(prior to 1940) the ﬁrst major concept, that of a membrane enclosing the cell contents was born from permeability studies dating back to the second half of 19 th
century, when it was observed3 that marine eggs and
RBCs behaved as osmometers: the cells would swellin dilute media and shrink in concentrated media. The
conclusion was that the cells are surrounded by amembrane allowing water exchanges between cell andmedium.
The second major concept was the quantitative deﬁnition of permeability as a permeability coefﬁcient (resistance) using diffusion equation (derivation of the
Fick’s equation). Overton4 studying the movement ofwater and nonelectrolytes across cell membranes demonstrated that the most important determinant of permeability was the lipid solubility of the solute; however,since small hydrophilic solutes could also penetrate themembrane in an inverse relationship to molecular size,
he proposed a mosaic membrane largely lipid but withaqueous patches.
The third major concept of the ‘‘Early Period’’ wasthe proposition by Gorter and Grendel,5 from studies
of RBCs, that the plasma membrane consists of lipid
structure in a bilayer arrangement. Ten years later
studies on ﬁsh eggs led Danielli and Davson6 to propose that the permeability barrier of the cell surface
was a lipid bilayer sandwiched between protein monolayers.
In the ‘‘Classical Membraneology Period’’ (1940–1970) studies of transport held center stage owing to theIntroduction: Deﬁnition of Water Channels
Water transport in cells and tissues is essential for
life. When water molecules move into or out of the cellor from one subcellular compartment to another they
have to cross the cell membranes, which form the barrier between the interior and exterior of cell or between
cell organelles and cytosol. I have edited a two volume
book1 providing in depth presentations of water transport in a variety of biological membranes.
Since the membrane proteins confer to biologicalmembranes a much higher water permeability compared to the lipid counterpart, by water channels weunderstand in fact the water channel proteins, thosetransmembrane proteins that have as their main (speciﬁc) function the transport of water. In 1993 the water
channel proteins were called aquaporins.
Evolution of Concepts on Water Transport acrossBiological Membranes
The evolution of concepts on water transport acrossbiological membranes matches the advances of membraneology, that can be divided2 into four periods: the‘‘Early Period’’ (prior to 1940), the ‘‘Classical Membraneology’’ (the period between 1940 and 1970), the‘‘Modern Membraneology’’ (1970–1984) and the ‘‘Molecular Membraneology’’ (since 1985).
The red blood cell (RBC) appeared to be ideallysuited for investigating water permeability because of
its availability and simple structure, lacking internalThis title is the same as the lecture which Dr. Benga gave at the 1421st Meeting of The Keio Medical Society on June 10, 2005.
Reprint requests to: Dr. Gheorghe Benga, Professor and Chairman, Department of Cell and Molecular Biology, ‘‘Iuliu Hat,ieganu’’ Universityof Medicine and Pharmacy Cluj-Napoca, 6 Pasteur St., Cluj-Napoca 400076, Romania, e-mail: gbenga@umfcluj.ro; gbenga@personal.ro64

Keio J Med 2006; 55 (2): 64–69

availability of radioactive isotopes which allowed easy
and accurate measurements of ﬂuxes of ions and molecules and also the use of inhibitors or ‘‘chemical probes’’
that could perturb speciﬁc transport systems. Stein andDanielli7 proposed that hydrophilic pores in the membrane could account for the movement of water andions.
Solomon and coworkers (after 1950) introduced thetechniques to measure the RBC osmotic water permeability (named also the ﬁltration or hydraulic conductivity, measured by the net ﬂux of water in response toa hydrostatic or osmotic pressure gradient), and the
RBC diffusional water permeability.8,9 Since in thecase of RBC the osmotic permeability coefﬁcient ðPf Þ is
2.5 times greater that the diffusional permeability coefﬁcient ðPd Þ, while in lipid bilayers the ratio is unity (inaccord with expectations for a nonporous structure) the
difference between the two rates of water transport in
RBC has been interpreted as indicating the presence ofwater-ﬁlled channels or pores in the membrane. An estimate of the ‘‘equivalent radius’’ of these channels (onthe assumption of uniform right cylindrical pores) was
performed employing hydrodynamic principles from theratio of the Pf and Pd . The ‘‘equivalent radius’’ of thewater channels was ﬁrst calculated as equal to 3.5 Å,and the effective area of pores in the human RBC wasestimated to lie between 0.01–1% of the total surface.8
Unfortunately, Solomon changed later several times his
estimation giving at some time values of 4.5 Å for theequivalent pore radius in the human RBC and 6.1 Å in
dog RBC and ﬁnally concluded that water crosses thehuman RBC membrane through an equivalent pore
whose radius of 6.5 Å is large enough to permit thepassage of ions and nonelectrolytes.10
Macey and Farmer11 found that incubation of RBCs
with the sulfhydryl (SH) reagents p-chloromercuribenzoate (PCMB) and p-chloromercuribenzene sulfonate (PCMBS) can produce a dramatic decrease (up to90%) in RBC osmotic water permeability and this
could be reversed by cysteine. As the maximal inhibitory effect of mercurials was to reduce the osmotic
water permeability by a factor of 10, i.e. to a value
similar to the diffusional permeabilities obtained withlecithin-cholesterol bilayers, Macey and Farmer11 suggested that ‘‘the action of PCMB and PCMBS may be
to inhibit water ﬂow through aqueous channels (pores)
leaving the lipid portions of the membrane as the only
alternative for water transport’’. There was no idea
where the water channels were localized in the RBC
membrane and which of the membrane proteins could
accommodate these channels.
In the ‘‘Modern Membraneology Period’’ (1970–
1984) a series of technological advances have been followed by rapid exploitation (see various chapters in
Benga12): the formulation of the ﬂuid mosaic model for65

the structure of biological membranes; the concept oftransmembrane proteins and the beginning of understanding of their functional architecture; the use of‘‘probes’’ to ‘‘mark’’ functional proteins that lead to theidentiﬁcation of some transport proteins (such as band
3 protein, the anion transporter in the RBC membrane); sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) for separating and identifying membrane proteins; puriﬁcation of transporters
and their functional reconstitution in model membranes
and others.
The RBC continued to be one of the most favoredcells for studies of water permeability to this preferreduse contributing the fact that the rather detailed
knowledge of molecular structure of RBC membrane
became available, regarding both the lipid and the protein counterpart.13
On the other hand new methods for measuring water
permeability became available. In 1976 together with
Vasile Morariu we began systematic studies of the human RBC water permeability, by using a nuclear magnetic resonance (NMR) technique described by Conlon
and Outhred.14 This method was evaluated by us15–17
and then I used it extensively to characterize various
aspects of water permeability of human RBC andresealed ghosts, not only during this period, but also inthe ‘‘Molecular Membraneology Period’’ (since 1985).
We have shown for the ﬁrst time by NMR that theparameters characterizing water permeability such as
Pd , and the activation energy ðEa; d Þ, are the same in
RBCs and resealed ghosts18–21 and have reported thelargest series of determinations of water diffusional
permeability of RBCs available in the literature.22 We
have studied in detail the effects of various inhibitorsof water permeability,23–27 uncovering new ﬁndings,
such as the irreversible inhibition induced by ﬂuoresceinmercuriacetate or the powerful inhibition
induced by mersalyl. We have also shown that water
permeability of RBCs is not affected by proteolytic
enzymes, i.e. that the RBC water channels proteins areimpervious to proteolytic digestion.28 In addition, theenzymic treatment of membranes, or the preincubation
with SH reagents that are not inhibitors of water permeability, such as N-ethyl-maleimide (NEM) or iodoacetamide (IAM), did not prevent the inhibition inducedby mercurials. No ultrastructural changes could be seen
by electron microscopy after incubation of RBCs withSH reagents.29
In this period it was accepted that the water transport through the RBC membrane involves two parallelpaths: a) through the water channels formed by integral
membrane proteins, and b) via diffusion through thebackground lipid bilayer. The inhibition of water transport by SH group blocking reagents was interpreted as
an indication of water channel blockage. Consequently,

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Benga G: Discovery of water channel proteins (Aquaporins)

experiments aimed at associating water channels withspeciﬁc membrane proteins using radioactive-sulfhydryllabeling methods became feasible. Brown et al.30 werethe ﬁrst to report the results of labeling experiments
using 14 C-DTNB (dithiobisnitrobenzoate) after preincubation of the cells with N-ethyl-maleimide (NEM)
and iodoacetamide (IAM). A binding of 14 C-DTNB to
the band 3 protein was found and they suggested thatband 3 is involved in water transport, on the assumptionthat DTNB is an inhibitor of this process. However, nomarked inhibition of water diffusion could be induced
by DTNB,24,31 so this experiment is not relevant. Laterwork by Sha’aﬁ and Feinstein32 presented evidence forselective labeling of band 3 with 14 C-PCMBS after preincubation of RBCs with IAM, NEM and mersalyl,
compounds that were considered not to inhibit transport. However, we demonstrated that mersalyl is astrong inhibitor of diffusional water permeability,24 consequently this labeling experiment is also not relevant.
In 1982 I organized together with Fred Kummerow(Burnsides Research Laboratory, University of Illinois
at Urbana-Champaign) a Romanian-American Workshop in New York City and the proceedings were published next year by The New York Academy of
Sciences. At the Workshop Solomon reported the localization of [ 203 Hg]-PCMBS on band 3 following incubation with human RBC ghosts at 0 C for 2 min, at 0.1
mM ﬁnal concentration.33 However, the inhibition ofwater permeability was not estimated. Solomon, Verkman and coworkers33 continued to claim that no speciﬁc water channel exists and that a protein migrating asband 3 on the electophoretogram of RBC membranes
is a common pore for water, cations, anions and nonelectrolytes.
After my return to Cluj-Napoca we found that longer
incubation times or higher temperatures are needed inorder to induce the inhibition of water transport.18
In conclusion none of these labeling experiments
provided clear indication which of the RBC membraneproteins could accommodate the water channel.
The First Discovery of a Water Channel Protein
I believe that everybody agrees with the following
deﬁnition of discovery: ‘‘to discover’’ means ‘‘to learn
or see for ﬁrst time’’.34
In 1985 my group performed the labeling experimentby which the presence and location of a water channel
protein in the human RBC membrane was clearly
demonstrated for the ﬁrst time; the results were published in 1986.35 The water channel protein was identiﬁed among polypeptides migrating in the region of 35–
60 kDa on the electrophoretogram of RBC membranes,
labeled with [ 203 Hg]-p-chloromercuribenzene sulfonate(PCMBS) under conditions of speciﬁc inhibition ofwater diffusion. It is worth discussing in detail the experimental conditions that allowed us to obtain a very
selective labeling of the water channel protein as illustrated in Figure 2 (a) of our landmark publication.35
Resealed ghosts, suspended at a cytocrit of 25% in
an isotonic medium (150 mM NaCl, 5 mM sodium
phosphate buffer, pH 7.5), were preincubated with 2
mM N-ethyl maleimide (NEM) for 60 min at 2 C. In
this way we blocked almost all SH groups that are not
involved in water transport. After the preincubation theghosts were diluted with the same isotonic medium
containing 2 mM NEM at 10% and incubated with 0.1
mM [ 203 Hg]–PCMBS (the inhibitor of water transport),for 15 min at 0 C or for 5 min at 37 C.
NMR measurements of water permeability showedthat at 0 C there was no inhibition of water permeability; 5 min at 37 C is the minimum time required to see asigniﬁcant inhibition. After the incubation, resealed
ghosts were washed three times in 20 volumes of ahypotonic medium (50 mM NaCl, 4 mM sodium phosphate, pH 7.5, 2 mM NEM), by centrifugation at 8000
g for 10 min. at 4 C. Puriﬁed membranes were preparedfrom resealed ghosts to remove 203 Hg-PCMBS that may
have bound to haemoglobin and other cytoplasmic
components. Membrane polypeptides were separated
by electrophoresis, the gel cut into 2 mm slices and theradioactivity measured. The protein binding PCMBS
under these conditions were identiﬁed by superpositionof the radioactivity on the densitometric tracing
obtained by scanning gels stained with Commasie Blue.
The nomenclature devised by Fairbanks et al.13 wasused to identify the membrane proteins.
Under conditions where marked inhibition of watertransport was ﬁrst obtained (after the incubation at
37 C) the inhibitor was bound to the membrane proteins migrating as band 3 and band 4.5 (actually thepolypeptides migrating in the region of 35–60 kDa on
the electrophoretogram, as can be seen in Fig. 2 (a) in
our landmark publication.35 This binding pattern suggested that either or both band 3 and 4.5 proteins could
be associated with water channels. However, we pointed
out that polypeptides migrating in this regions havealready been identiﬁed in other transport functions,notably anion exchange (band 3 protein) and thetransport of glucose and nucleosides (band 4.5 protein).
There was no evidence that a speciﬁc inhibitor of one ofthese processes will inhibit water transport.35
Consequently, I concluded:35 ‘‘It remains possiblethat a minor membrane protein that binds PCMBS is
involved in water transport’’, since the only way to explain our results was to postulate that PCMBS is boundto a protein in the RBC membrane that has not beenpreviously seen. I also indicated the way in which thespeciﬁc protein could be further characterized: by puriﬁcation and reconstitution in liposomes.

Keio J Med 2006; 55 (2): 64–69

In the same year the labeling experiments wereconﬁrmed and extended.36 In the following 2–3 years
I described the novelty of our work in several
reviews.37–40 It should be emphasized that our ﬁrst
landmark paper35 was published in a well known
American journal, the invited reviews were publishedin well known international series37–39 and, moreover, I
reviewed again our work40 in the second volume of thebook that I edited for CRC Press, Boca Raton.1
In 1988, Agre and coworkers puriﬁed a new protein from the RBC membrane,41 nicknamed CHIP28(channel-forming integral membrane protein of 28kDa).42 However, in addition to the 28 kDa component, the protein had a 35–60 kDa glycosylated component, i.e., the one we detected as the binding site of
PCMBS under conditions for the inhibition of watertransport across the RBC membrane.35,36 They suggested that CHIP28 may play a role in the linkage ofthe membrane skeleton to the lipid bilayer.41
In 1990, Parker ﬁrst suggested in personal discussion
to Agre that the novel protein might be the water
channel, and in 1992 Agre and coworkers43 based on
Windager’s suggestion to use oocyte expression as amechanism to study water transporters, found thatoocytes from Xenopus laevis microinjected with invitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability. The water permeability was
inhibited by mercuric chloride, therefore, it was suggested that CHIP28 is a functional unit of membranewater channels. By reconstitution in liposomes it was
shown that CHIP28 is a water channel itself rather
than a water channel regulator. In 1993 CHIP28 wasrenamed aquaporin 1.44
Since 1993 water channel proteins became a very hot
area of research; more than 200 members of the aquaporin family have been found in bacteria, plants, animals and humans; a great diversity of physiological andpathological implications are being uncovered. In 2003
Peter Agre was awarded the Nobel Prize in Chemistry‘‘for the discovery of water channels’’.45
It is obvious and overwhelmingly documented fromthe facts presented above that the ﬁrst water channel
protein (aquaporin 1) was ﬁrst discovered by myself
and my coworkers in 1985 in Cluj-Napoca, Romania,
and reported in publications in 1986.35,36 We identiﬁed
its glycosylated component with a molecular weight of
35–60 kDa and indicated the way to distinguish it fromother proteins (reconstitution in liposomes and measurement of water permeability). Aquaporin 1 was ﬁrst
puriﬁed in 198841 and its water transport properties
were identiﬁed in 1992 by Agre and coworkers.43 It isalso obvious that what we identiﬁed by labeling experiments is the same protein that Agre and coworkers
later puriﬁed, since they mentioned43 that ‘‘the characteristics of CHIP28 are consistent with other known

67

features of water channels, e.g. CHIP28 proteins in intact RBCs are impervious to proteolytic digestion, as
are water channels’’, citing ref.28
As Agre and coworkers cited our 1983 paper28 it isvery surprising that they never cited our landmark 1986
papers;35,36 in contrast they referred only to work byother American scientists who pointed to a non-speciﬁc‘‘pore’’ that allowed permeation of anions, cations,nonelectrolytes and water.33 In contrast, we strongly
argued all the time that there were indeed water channels in the RBC membrane and indicated the way
how speciﬁc water channel proteins could be further
characterized by puriﬁcation and reconstitution in liposomes.
Recently, the Nobel Lecture of Agre was published.46 Although he cited Benga twice among ‘‘pioneers in the water transport ﬁeld’’ none of Benga’s
publications were listed in References.
I continued to be very active in the ﬁeld, and published many papers including one on the puriﬁcation ofaquaporin 1 and also developing a new procedure forits quantiﬁcation by densitometry of silver stained gel.47
Over the last decade, we have characterized the water
permeability of RBCs from over 30 species, reviewed in
ref.48,49 We reported a positive correlation between thewater permeability values of RBCs from maternal venous blood and fetal RBCs isolated from cord bloodtaken at delivery; this points to a genetic basis for thedetermination of RBC water permeability.50
Looking back over years let’s ask the crucial question, when was the ﬁrst water channel protein, aquaporin 1, discovered? If a comparison with the discoveryof The New World of America is made, the ﬁrst man
who has ‘‘seen’’ a part, very small indeed, of The New
Land was Columbus; later, others, including Amerigo
Vespucci (from whom the name derived), have better‘‘seen’’ a larger part of the new Continent and in thesubsequent years many explorers discovered the complexity of the Americas!
‘‘No one had seen this protein before’’ said Agre.45
However, this protein was ﬁrst seen by my group in
Cluj-Napoca, Romania, in 1985, reported in publicationsin 198635,36 and reviewed in subsequent years.37–40
Consequently, a fair and clear cut answer to the above
mentioned question would be: the ﬁrst water channel
protein, now called aquaporin 1, was identiﬁed or‘‘seen’’ in situ in the human RBC membrane (hence
discovered) by Benga and coworkers in 1985. It wasagain ‘‘seen’’ when it was by chance puriﬁed by Agreand coworkers in 1988 and it was again identiﬁed(hence re-discovered) when its main feature, the watertransport property, was found by Agre and coworkers
in 1992. It appears that our seminal contributions published in 1986 were grossly overlooked by Peter Agreand by the Nobel Prize Committee.

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Benga G: Discovery of water channel proteins (Aquaporins)

I presented the above mentioned claim in a PETITION on October 18th, 2003 at The 8th World Congress on Advances in Oncology, and 6th International
Symposium on Molecular Medicine (Hersonissos, Crete,
Greece) and immediately scientists from a lot of countries have signed in support of my PETITION. Afterthe PETITION and additional information was placedon the web site of the Ad Astra Association (www.ad-astra.ro/benga) thousands of science-related professionals from hundreds of academic and research
units, as well as participants in several international
scientiﬁc events, have signed as supporters of my priority, that is also mentioned in comments on the 2003
Nobel Prize in Chemistry.51 I have also received many
messages from scientists who considered that I was theﬁrst discoverer of the ﬁrst water channel protein andthat my omission from the Nobel Prize is a mistake. In
addition the above mentioned facts were presented andacknowledged at over 25 international scientiﬁc eventsin 2004 and 2005, including two world conferences, aswell as in seminars at many European, American andJapanese universities in 2003–2005, steering favorable
reactions; as can be seen on the above mentioned site
the recognition of Gheorghe Benga as a discoverer ofthe ﬁrst water channel protein from the human RBC
membrane is growing.
Acknowledgements: I acknowledge the collaboration of all my coworkers over the past 25 years (coauthors in publications listed
below). The ﬁnancial support of The Ministry of Education, Academyof Medical Sciences, National Council for Science and Technology(Romania), The Wellcome Trust, The British Council (UK) and The
National Science Foundation (USA) for the work reviewed in thispaper is gratefully acknowledged. I also thank Professor Nobuyoshi
Shimizu and The Keio Journal of Medicine for sponsoring my seminar
at Keio Medical School in June 2005.

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