Molecular Physiology and Evolution
of Membrane Transport Systems
To challenge Entropy Life
recruits an array of membrane mechanisms including ATPase pumps,
ion channels, and secondary transporters which act in synergy to
generate and manage transmembrane solute gradients. The secondary
transporters evolved variety of mechanisms to maintain cellular
homeostasis by coupling electrochemical gradients of inorganic
ions, substrates, and metabolites. Our objective is to determine
the molecular, integrative, and evolutionary basis of secondary
transporters which supply essential minerals and nutrients in organisms.
We employ simple comparative models with known genome sequences
and explicit biological plans including the African malaria mosquito Anopheles
gambiae , yellow fever mosquito Aedes aegypti ,
and fruit fly Drosophila melanogaster which comprise a
time-cost-efficient experimental framework to study molecular,
electrochemical, and integrative basis of the transport systems.
Our research leads to the identification, functional characterization,
and structural homology modeling of novel transporters. Ultimately
we plan to establish an interactive bioinformatics platform for
molecular modeling, engineering, and managing of transport systems
in medical, pharmacological, and biological applications.
Current
Projects
Identification
and characterization of new
amino acid transporters
(supported by NIH-NIAID -R01 research grant AI30464).
Our primary focus is on the Sodium Neurotransmitter
symporter Family,
SNF (a.k.a. SLC6). This large family includes transporters for neurotransmitters,
amino acid osmolites and energy sources, and recently identified subfamily of Nutrient
Amino acid Transporters (NATs; Boudko et al. PNAS, 2005). Molecular
cloning and functional characterization of new insect NATs in our laboratory
led to the discovery of unique transport phenotypes for specific subsets of amino
acids. We showed that NATs constitute a previously-elusive synergetic transport
mechanism for absorption and distribution of essential amino acids which organisms
are unable to synthesize. Hence, NATs are critical for essential amino acid balance
and metabolism, development and brain function; NAT dysfunction induces metabolic
and mental disorders including anxiety, drug abuse, obesity, and distinct forms
of neutral amino acidurias; and species-specific NAT function is faultless target
for and environmentally-safe biological control of pathogenic and pest organisms.
Our present studies aim to explore molecular integrative basis and structure-function
relationship in the NAT population. Understanding evolution of NAT structure
and function will diploy a new dimension for in silico prediction, simulation,
and molecular engineering of new SNF phenotypes. It also will facilitate drugs
discovery to correct metabolic and neuronal disorders and suppress eukaryotic
pathogens. To understand NATs we aim to clone, characterize and compare NAT populations
which are present in genomes of biomedically important vector mosquitoes and
fruit fly. Presently we have cloned 90% of NATs from the selected dipterans. A
number
of these NATs were functionally
expressed in Xenopus oocytes
and characterized using electrochemical
analysis, uptake of
isotope-modified substrates, whole-mount in
situ hybridization and immunolabeling with
epitope-specific antibodies. We discovery that NATs population expresses array
of unique transport mechanisms with specific substrate profiles but complementary
function in epithelial absorption and somatic redistribution of all essential
amino acids and some related metabolites. The NAT function in the alimentary
canal is regulated via alternative spatial expression and polarized docking of
NATs in secretory and absorptive epithelia. We also identified correlations of
aromatic NATs with monoamine neurotransmitters in the brain, and concentration-dependent
shift of substrate-coupling stoichiometry. The future analysis of these phenomena
will substantially improve our knowledge of molecular and integrative mechanisms
of absorption, balance, and metabolism of essential amino acids and neurotransmitters
in insects as well as other organisms, including humans.
 |
Phylogenetic
tree of Sodium Neurotransmitter symporters Family (SNF)
in selected model organisms. Yellow background
outlines complete genomes, gray and red arrows respectively
represent cloned and characterized insect NATs. Abbreviations:
DA, 5HT and GABA indicate orthologous groups of catecholamine,
serotonin and GABA NeuroTransmitter Transporters (NTTs),
B0 and B0+ are broad substrate spectra transporters for
neutral and neutral + cationic amino acids, respectively. Click
on image for larger view. |
Transport Physiology of Disease Vector Mosquitoes (in collaboration with
Drs. W. Harvey and P.
Linser)
This project focuses on the analysis of cationic and anionic mechanisms which
produce ultimate alkalinization (pH>11) in the anterior midgut of mosquito
larvae. We have developed a semi-intact
larvae preparation that is suitable for monitoring of electrophysiological,
microchemical, and ion flux parameters of individual epithelial cells under virtually
natural conditions. Analysis of such preparations revealed that the anterior-specific
lumen alkalinization in the larval midgut (pH11) is generated via transepithelial
anion and cation exchangers which in mosquito larvae energize via the electrogenic
H + V-ATPase pump (JEB & PNAS 2001). Currently we employ semi intact preparations
to define electrophysiological, pharmacological, and ionic flux in
specified midgut regions and epithelial cells. We analyze transmembrane potential,
spatial electrochemical coupling, and local ionic fluxes in different midgut
regions under model physiological conditions and applications of specific ion
inhibitors. Presently we have cloned several anion and cation exchangers from
SLC4 and CLC9 families of An. gambiae. We have proved that several these
transporters are associated with alkalinization. Future analysis will focus on
a comprehensive understanding of mineral ion homeostasis and will disclose the
molecular and integrative basis of the essential alkalinization phenomenon.
Microchemical
analysis of single epithelial
cell.
To
understand the physiology and
biophysics of membrane transport
systems in vivo we need to know
the distribution of electrochemical
gradients and ion fluxes in
the epithelial tissues. Several
new approaches were successfully
deployed in our laboratory including
the capillary electrophoresis
(CE)
of subpicoliter biological samples
(~ 10^-12 liter) and Self-Referencing
Ion-Selective microelectrodes
with Liquid Ion eXchanger (SERIS
- LIX a.k.a SIET or Scanning
Ion-selective Electrode Technique)
which allows profiling of chemical
compositions and transmembrane
movements of organic and inorganic
solutes with subcellular resolution.
We continue developing and optimizing
new micro-analytical protocols
and integrating single cell
analytical assays with conventional
electrophysiological techniques
e.g. intracellular microelectrodes,
voltage clamp, patch clamp,
and heterologous molecular expression.
New
Projects
Exploring NATs for control of vector mosquitoes
We
have started to analyze the potential impact and selectivity
of pharmacological suppression and RNAi silencing of essential
NATs in disease vector mosquitoes. We are also studying the regulatory
epigenetics of essential amino acid transporters in distinct tissues
and developmental stages of mosquitoes and plan to explore the capacity
of epigenetic inheritance in the NAT population upon nutrient and
mineral stress. These data will be used to support the competitive
continuation of my NIH R01 AI52436 grant.
Sequence-structure-function
relationships model in the SNF.
The crystal
structure of a NAT from the bacterium Aquifex
aeolicus VF5 (2A65) has been solved ( Yamashita et al.,
Nature 2005). Insect and bacterial NATs share ~20% sequence identities
with a much stronger conservation in an substrate binding region
(> 50%), thus satisfying a homology modeling of these transporters
with a focus on a substrate binding envelopes. Identification
and characterization of new NAT phenotypes in combination with in
silico structural modeling provide a unique possibility
to improve understanding of substrate binding and conformational
models of SNF as well as to trace mutations which lead substrate
and electrochemical adaptation of NAT phenotypes. Currently we
have built an array of three-dimensional models of SNF members
based on reciprocal structural homology and homology with a 2A65
(Fig. 1). Using these data I plan to compare key structural properties
of NAT members which determining substrates specificity as well
as substrate coordination and coupling efficiencies. The 3D models
can also be used for de novo prediction of substrate
profiles and high-throughput in silico docking of
ligand libraries with aim to identify potent modulators of NAT
functions.
Role of NATs in neurochemical
signaling
Nutrient
amino acid transporters and neurotransmitter transporters of
the SLC 6 family share a common ancestor and structural properties.
We found that neuronal NATs are colocalized with
monoamine synthesis pathways. It is possible that some antidepressant
drugs which are used to suppress monoamine absorption in synapses
via monoamine uptake transporters (e.g. DAT, SERT, and NET) could
also impact the absorption of essential precursors for the synthesis
of monoamine neurotransmitters via NATs. This could ultimately compromise
the endogenous synthesis of neurotransmitters. I would like to address
these issues using heterologous expression and computational modeling
of mammalian and insect NATs as well as in vivo and in
vitro models of neurotransmitter synthesis pathways.
Molecular reconstruction of ancestral SNF members
The evolution
of complex functions and structures in membrane proteins is a fundamental
question of molecular physiology. This problem is difficult to
access experimentally and has so far only been amenable to in
silico simulation. Nevertheless, reconstruction of
ancestral forms was successful for some proteins, such as pigments
and GFPs. In collaboration with Dr. M. Matz we
launched a new project for phylogenetic prediction and molecular
reconstruction of transporters which would represent key nodes in
the evolutionary tree of the SNF. We hope to acquire confident information
about the electrochemical properties and substrate spectra of ancestral
NATs. In addition, these studies will provide information about evolutionary
mechanisms and structural constraints which drive diversification
and plasticity of NATs and other SNF members.
Molecular and structural analysis of accumulative
membrane transport of vitamins
We have cloned the first invertebrate
ascorbic acid transporters (NCBI AAM97679 and AAM97678 ) and soluble
vitamin transporters (NCBI AAL38977) respectively from the malaria
and yellow fever vector mosquito. Both An. gambiae and Ae.
aegypti larvae
are capable of concentrating up to 10 mM ascorbate / dehydroascorbate
in anterior midgut cells and lumen (based on capillary electrophoresis
of microsamples from mosquito larvae). We anticipate that interference
with ascorbate recycling transporters would impact critical functions
of the larval alimentary canal including midgut alkalinization,
free radical tolerance, and pathogen defenses.
Development of a high-throughput platform for heterologous expression
and characterization of membrane transport proteins
Pharmacological
screening and characterization of new transporter phenotypes has
high priority. In collaboration with Dr.
Peter J. S. Smith ( Woods Hole BioCurrents Research Center)
we plan to develop a multi-channel microsensor array system which
will combine standard electrophysiological recording with organic
and inorganic substrate flux monitoring under conditions of heterologous
expression of transport proteins. The combination of the noninvasive
self referencing ion-selective and electrochemical microelectrodes
with heterologous expression promises to open new avenues for the
characterization of transport mechanisms, including superior resolution
over a generic voltage-clamp and the capacity to record multiple
fluxes as well as electro-neutral events. Sensors for electrochemical
detection of amino acids are under development. In addition, such
assays can replace expensive and time-consuming isotope uptake
methods and would allow real-time monitoring of substrate-coupling
stoichiometry.
A
simplified diagram of Noninvasive Microelectrode Array
scanning System (NMAS).
Two microelectrodes:ISM,ion
selective microelectrode (e.g. Na + selective) and ESM, electrochemical
sensing microelectrode (e.g. amino acid selective) are shown.
MM1, is micromanipulator for alignment of the probes, MM2 is
precision motion control device. +1 and ∞ labels represent
a potentiometric and amperometric head stage amplifiers respectively. Σ labels
differential amplifiers. XEO is Xenopus oocyte in
a microperfusion chamber. Interface connector references are:
1. Ion flux; 2, flux stoichiometry; 3, amino acid flux, 4,
an electrochemical probe polarization signal. |
Personnnel
Dmitri
Y Boudko, Research Assistant
Professor
William R. Harvey, Professor
of Physiology and Functional
Genomics, Co-PI
Melissa Miller, Postdoctoral
associate
Bernard Okech, Postdoctoral
associate
Ella A. Meleshkevitch, Senior
biologist
Lyudmila Popova, Visiting researcher
Dmitri Voronov, Visiting researcher
Current
Research Support
NIH
R01, 2004 – 09
Principal Investigator of AI30464
NIH-NIAID grant. Physiology
of Insect Amino Acid Transport.
The goals are to clone and characterize
essential amino acid transporters
from larval midgut of malaria
vector, Anopheles gambiae.
NIH,
R01 2003 - 08 Co-principal
Investigator of AI52436 NIH-NIAID
grant. Transport Physiology
of Disease Vector Mosquitoes.
The goal is to understand the
role of H+ V-ATPases and cation
exchangers in alkalinization
of the insect gut and mineral
ion homeostasis (PI, W.
Harvey).
Selected
Publications
Boudko D.Y. (In press) Bioanalytical
profile of L-arginine/nitric oxide pathway and its analysis by
capillary electrophoresis .J. Chromatography
B.
Meleshkevitch
EA, Assis-Nascimento P, Popova LB, Miller MM, Kohn AB, Phung
EN, Mandal A, Harvey WR, Boudko DY. (2006) Molecular
characterization of the first aromatic nutrient transporter
from the sodium neurotransmitter symporter family. J
Exp Biol. Aug;209(Pt 16):3183-98. (PDF)
Boudko
DY, Kohn AB, Meleshkevitch EA,
Dasher MK, Stevens BR. and Harvey
WR. (2005) Ancestry and Progeny of
Nutrient Transporters. PNAS
102(5):1360-5 (PDF).
Boudko DY, Stevens BR, Donly
B C. & Harvey WR. (2005) Nutrient
Amino acid and Neurotransmitter
transporters, In: Comprehensive
Molecular Insect Science. First
edition. Editors: Lawrence I.
Gilbert, Kostas Iatrou and Sarjeet
S. Gill. Elsevier, Amsterdam. V.4, 255-309.
Moroz
LL, Dahlgren RL, Boudko D, Sweedler
JV & Lovell P. (2005) Direct single
cell determination of nitric
oxide synthase related metabolites
in identified nitrergic neurons.
J Inorg Biochem 99(4) 929-39.
Boudko
DY. (2004) High resolution capillary
electrophoresis of nitrite and
nitrate in biological samples.
In: Methods in Molecular Biology;
Nitric Oxide Protocols, Second
Edition. Editors: Aviv Hassid,
Humana Press Totowa, NJ, USA.
V.100, 9-19.
Boudko
DY, Yu HS, Ruiz M, Hou S, Alam
M. (2003) A time-lapse capillary assay
to study aerotaxis in the archaeon
Halobacterium salinarum. J Microbiol
Methods 53: 123-126.
Croll
RP, Boudko DY, Pires A, Hadfield
MG. (2003) Transmitter contents of
cells and fibers in the cephalic
sensory organs of the gastropod
mollusc Phestilla sibogae. Cell
Tiss Res 314:437–448.
(PDF)
Boudko
DY, Cooper BY, Harvey WR &
Moroz, L.L. (2002) High-resolution
microanalysis of nitrite and
nitrate in neuronal tissues
by capillary electrophoresis
with conductivity detection.
J Chromatogr 774(1): 97-104.
(PDF)
Boudko
DY, Moroz LL, Harvey WR, &
Linser PJ. (2001) Alkalinization by
chloride/bicarbonate pathway
in larval mosquito midgut. PNAS 98: 15354-15359. (PDF)
Boudko
DY, Moroz LL, Linser PJ, Trimarchi
JR, Smith P JS. & Harvey
WR. (2001) In situ analysis of pH gradients
in mosquito larvae using non-invasive,
self-referencing, pH-sensitive
microelectrodes. J Exp Biol 204: 691-699. (PDF)
Croll
RP, Boudko DY & Hadfield
MG. (2001) Histochemical survey of
transmitters in the central
ganglia of the gastropod mollusc
Phestilla sibogae. Cell Tiss
Res 305: 417–432.
Hou
S, Larsen RW, Boudko D, Riley
CW, Karatan E, Zimmer M, Ordal
GW & Alam M. (2000) Myoglobin-like
aerotaxis transducers in Archaea
and Bacteria. Nature 304:
540-544.
Hadfield
MG, Meleshkevitch EA & Boudko
DY. (2000) The apical sensory organ
of a gastropod veliger is a
receptor for settlement cues.
Biol Bulletin 198: 1, 67-76.
Boudko
DY, Switzer-Dunlap M. &
Hadfield MG. (1999) Cellular and subcellular
structure of anterior sensory
pathways in Phestilla sibogae
(Gastropoda, Nudibranchia). J Comp Neurol 403: 39-52.
Budko
DY, Moroz LL & Gourine VN.
(1998) Simple apparatus for the electrical
clearing of glass microelectrodes.
Neurosci Behav Physiol 28: 86-89.
Budko DY, Moroz LL, Winlow W
& Gourene VN. (1994) Thermocontroller
for neurophysiological studies.
Thermophysiology 4: 59-63.
Based
on the heterologous activities and expression profiles we propose
that NATs represent an active core of the transport system for
absorption and redistribution of essential amino acids. Phylogenetic
analysis suggests that the first universal ancestor of NATs and
SNF emerged in protobacteria. NATs represent a set of lineage-specific
expansion in the evolutionary tree of the SNF and triggered a
chain of dramatic events in the history of life. First, it compromised
nitrogen fixation and derived the virtual extinction of this
energetically expensive pathway in eubacteria. Secondary NAT
expansion and specialization in metazoan ancestors compromised
intracellular synthesis of essential amino acids which trigger
the evolution of digestive, motor, and sensory systems as well
as a universal interface of those system, the brain. Some metabolites
of essential amino acids have been recruited as messengers of
chemical integration in multicellular organisms. This sketchy
scenario perfectly reflects an image which is fossilized in a
contemporary phylogenetic tree and the hierarchy
of transport phenotypes of the SNF.
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