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Molecular and functional characterisation of a new mammalian family of proton-coupled amino acid transporters [Elektronische Ressource] / Martin Foltz

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Lehrstuhl für Ernährungsphysiologie Molecular and functional characterisation of a new mammalian family of proton-coupled amino acid transporters Martin Foltz Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. rer. nat. Michael Schemann Prüfer der Dissertation: 1. Univ.-Prof. Dr. rer. nat. Hannelore Daniel 2. Hochschuldozentin Dr. rer. nat. Ellen I. Closs, Johannes Gutenberg-Universität Mainz 3. Univ.-Prof. Dr. rer. nat. habil. Siegfried Scherer Die Dissertation wurde am 16.09.2004 bei der Technischen Universität München eingereicht und durch die Fakultät für Ernährung, Landnutzung und Umwelt am 15.12.2004 angenommen. iThe presented thesis is based on following peer-reviewed original papers and submittedmanuscriptsI Boll M, Foltz M, Rubio-Aliaga I, Kottra G, and Daniel H. Functional characterization oftwo novel mammalian electrogenic proton dependent amino acid cotransporters.J. Biol. Chem., 277, 22966-22973 (2002)II Boll M, Foltz M, Rubio-Aliaga I, and Daniel H. A cluster of proton/amino acid transportergenes in the human and mouse genomes.Genomics, 82, 47-56 (2003)# #III Boll M , Foltz M , Anderson CMH, Oechsler C, Kottra G, Thwaites DT, and Daniel H.

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Lehrstuhl für Ernährungsphysiologie


Molecular and functional characterisation of a new mammalian
family of proton-coupled amino acid transporters




Martin Foltz

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. rer. nat. Michael Schemann

Prüfer der Dissertation: 1. Univ.-Prof. Dr. rer. nat. Hannelore Daniel
2. Hochschuldozentin Dr. rer. nat. Ellen I. Closs,
Johannes Gutenberg-Universität Mainz
3. Univ.-Prof. Dr. rer. nat. habil. Siegfried Scherer



Die Dissertation wurde am 16.09.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät für Ernährung, Landnutzung und Umwelt am
15.12.2004 angenommen. i
The presented thesis is based on following peer-reviewed original papers and submitted
manuscripts
I Boll M, Foltz M, Rubio-Aliaga I, Kottra G, and Daniel H. Functional characterization of
two novel mammalian electrogenic proton dependent amino acid cotransporters.
J. Biol. Chem., 277, 22966-22973 (2002)
II Boll M, Foltz M, Rubio-Aliaga I, and Daniel H. A cluster of proton/amino acid transporter
genes in the human and mouse genomes.
Genomics, 82, 47-56 (2003)
# #III Boll M , Foltz M , Anderson CMH, Oechsler C, Kottra G, Thwaites DT, and Daniel H.
Substrate recognition by the mammalian proton-dependent amino acid transporter
PAT1.
#Mol. Membr. Biol., 20, 261-269 (2003) authors contributed equally
IV Rubio-Aliaga I, Boll M, Vogt-Weisenhorn DM, Foltz M, Kottra G, and Daniel H. The
proton/amino acid cotransporter PAT2 is expressed in neurons with a different
subcellular localization than its paralog PAT1.
J. Biol. Chem. 279, 2754-2760 (2004)
V Foltz M, Boll M, Raschka L, Kottra G, and Daniel H. A novel bifunctionality: PAT1 and
PAT2 mediate electrogenic proton/amino acid and electroneutral proton/fatty transport.
FASEB J. 18, 1758-60 (2004)
+VI Foltz M, Dietz V, Boll M, Kottra G, and Daniel H. Kinetics of bidirectional H and
substrate transport by the proton-dependent amino acid symporter PAT1.
Biochem. J. 384, accepted and in press (doi:10.1042/BJ20041519)
VII Foltz M, Oechseler V, Boll M, Kottra G, and Daniel H. Substrate specificity and transport
mode of the proton dependent amino acid transporter mPAT2.
Eur. J. Biochem. 271, 3340-3347 (2004)
VIII Anderson CMH, Grenade DS, Boll M, Foltz M, Wake KA, Kennedy DJ, Munck LK,
Miyauchi S, Taylor PM, Campbell FC, Munck BG, Daniel H, Ganapathy V, and Thwaites
DT. PAT1 (SLC36A1) is a nutrient and drug transporter in human and rat small intestine.
Gastroenterology 127: 1410-1422 (2004)iiTable of Contents
TABLE OF CONTENTS
SUMMARY 1
INTRODUCTION 3
1. Preface 4
2. Brief history of the discovery of amino acid transport processes 5
3. The amino acid/auxin permease family 8
4. Physiological importance of small, neutral amino acids and their derivatives
serving as substrates of the PAT-carriers 12
4.1 Glycine 12
4.2 Alanine 12
4.3 Proline 13
4.4 Amino acid derivatives and D-amino acids 13
5. Transport systems for small, neutral amino acids and amino acid derivatives 15
5.1 Glycine transporting systems 15
5.2 Alanine transporting systems 15
5.3 Proline transporting systems 17
5.4 Transport systems for GABA, betaine, taurine and D-amino acids 18
6. System PAT: Mammalian proton-coupled amino acid transport 20
7. Identification and cloning of a mammalian proton/amino acid symporter family 24
AIMS AND ACHIEVEMENTS OF THE THESIS 29
RESULTS AND DISCUSSION 33
1. Revelation of two new mammalian proton/amino acid transporters: General
characteristics 35
2. The PATs represent a new mammalian subgroup within the AAAP-family 37
3. Specific functional characteristics of PAT1 and PAT2 40
3.1 Minimal structural requirements of aliphatic amino acid PAT substrates 41
3.2 Driving forces for PAT-mediated transport 43
3.3 Bidirectional transport 44
3.4 Kinetic binding model 44
3.5 Lack of a proton-leak pathway 45Table of Content iii
4. PAT1 and PAT2 as potential delivery systems for short-chain fatty acids 47
5. Caenorhabditis elegans homologs of the mammalian PAT transporters 50
6. Proposed physiological and pharmacological roles of the PAT transporters 51
7. Potential pathological implications of PAT1 and PAT2 57
8. Future perspectives 59
ZUSAMMENFASSUNG 61
REFERENCES 65
ABBREVIATIONS 75
APPENDIX 1 77
APPENDIX 2 87
APPENDIX 3 99
APPENDIX 4 109
APPENDIX 5 117
APPENDIX 6 139
APPENDIX 7 171
APPENDIX 8 181
ACKNOWLEDGEMENT 195
CURRICULUM VITAE 199
LIST OF SCIENTIFIC PUBLICATIONS 201This thesis is dedicated to my parents
Barbara Foltz
and Gerhard Foltz
for their love and their encouragementSummary 1
SUMMARY
Proton-coupled amino acid transport (PAT) systems in the apical membrane of epithelial
cells of the small intestine and the renal tubule have been demonstrated by use of the human
intestinal cell line Caco-2 and renal brush border membrane vesicles. The present thesis
summarises the revelation of the molecular entity of the PAT system and provides deeper
insights into this new mammalian family of proton/amino acid cotransporters. The identified
family comprises four structural similar murine and human transport proteins (proton/amino
acid transporter, PAT1 - 4), all consisting of around 500 amino acid residues with 11
predicted transmembrane spanning domains. Orthologous proteins of this family are found in
lower and higher eukaryotes. The murine PATs exhibit a distinct mRNA expression pattern,
with PAT1 found inter alia in epithelial cells of small intestine and kidney. Moreover, the
PAT1 protein is localised in the brush border membrane of murine and human small
intestine. After expression in Xenopus laevis oocytes PAT1 mediates proton-coupled,
electrogenic transport of small and neutral amino acids such as Gly, Ala, and Pro, the
corresponding D-isomers and of a variety of amino acid derivatives, e.g. GABA, betaine, and
taurine. The apparent affinities for these substrates are in the range of 1 - 10 mM. Beside
these physiological substrates PAT1 transports efficiently selected pharmacological
compounds. As shown recently, PAT1 resembles in all features the functionally described
PAT system in intestinal and renal epithelial cells. The transporter PAT2, expressed in the
central nervous system, lung, heart, and brain exhibits similar transport characteristics as
PAT1. However, PAT2 is in comparison to PAT1 a high affinity transporter, with 10 to 30-
times higher affinities for the identical substrates (app. K -values of 100 - 1000 µM) but hasm
in general a narrower substrate spectrum. So far, PAT3 and PAT4 remain orphan
transporters with no assigned function. Critical structural elements in aliphatic amino acid
substrates of PAT1 and PAT2 were identified a) an non-substituted carboxyl-group, b) a
small size of the amino acid side chain, and c) a short spacer distance between the charged
amino and carboxyl groups. A free amino group within a substrate is not required.
Additionally, short-chain fatty acids were shown to serve as substrates of PAT1 and PAT2. In
contrast to amino acids, the short-chain fatty acids are transported in an electroneutral
manner. Both transporters seems to bind proton and substrate in an ordered binding mode
with the proton binding first. Functional differences in transport characteristics between PAT1
and PAT2 were observed in regard to the proton activation constants, suggesting that PAT2
transports at physiological pH values under proton-saturated conditions, whereas PAT1 can
be regulated in transport activity by changes in extracellular pH, i.e. via a cooperation with
+ +the Na /H -exchanger NHE3. This novel family of proton/amino acid symporters takes an
exceptional position within mammalian physiology.