New comprehensive biochemistry vol 21 molecular aspects of transport protein

MOLECULAR ASPECTS OF TRANSPORT PROTEINS


New Comprehensive Biochemistry

Volume 21

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Utrecht

ELSEVIER
Amsterdam . London . New York

Tokyo


Molecular Aspects of Transport
Proteins

Editor

J.J.H.H.M. DE PONT
Department of Biochemistry, University of Nijmegen,
6500 H B Nijmegen, The Netherlands

1992
ELSEVIER
Amsterdam . London . New York . Tokyo


Elsevier Science Publishers B.V.
P.O. Box 211
1000 AE Amsterdam
The Netherlands

L l b r a r y o f Congress Cataloging-ln-Publlcatinn

Data

M o l e c u l a r a s p e c t s o f t r a n s p o r t p r o t e i n s / e d i t o r . J.J.H.H.M. De P o n t .
p.
cm. -- (Nen c o m p r e h e n s i v e b i o c h e m i s t r y ; v . 2 1 )
I n c l u d e s b i b l i o g r a p h l c a l r e f e r e n c e s and i n d e x .
ISBN 0-444-89562-0
t a l k . paper)
2. I o n pumps--Molecular
1. C a r r i e r p r o t e i n s - - M o l e c u l a r
aspects.
3. I o n c h a n n e l s - - M o l e c u l a r a s p e c t s .
4. S o d i u n / p o t a s s i u m
aspects.
I.P o n t . J. J. H. H. M. de.
ATPase--Molecular a s p e c t s .
11. S e r i e s .
2. C a r r i e r P r o t e i n s [DNLM: 1. B i o l o g i c a l T r a n s p o r t - - p h y s i o l o g y .

-metabolism.
3. Membrane P r o t e i n s - - m e t a b o l i s m .
4. P r o t e i n B i n d i n g W1 NE372F V.21 / PU 55 M7145I
-physiology.
PD415.N48
vol. 21
[PP552.C341
5 7 4 . 1 9 ' 2 s--dc20
r574.87' 51
DNLMIDLC
92- 18220
for L i b r a r y o f Congress
CIP

ISBN 0 444 89562 0
ISBN 0 444 80303 3 (series)

0 1992 Elsevier Science Publishers B.V. All rights reserved
No part of this publication may be reproduced, stored in a retrieval system or transmitted in
any form or by any means, electronic, mechanical, photocopying, recording or otherwise,
without the prior permission of the Publisher, Elsevier Science Publishers B.V., Copyright &
Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands.
N o responsibility is assumed by the publisher for any injury and/or damage to persons or
property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because
of the rapid advances in the medical sciences, the publisher recommends that independent
verification of diagnoses and drug dosages should be made.

Special regulations f o r readers in the USA - This publication has been registered with the
Copyright Clearance Center Inc. (CCC) Salem, Massachusetts. Information can be obtained
from the CCC about conditions under which photocopies of parts of this publication may be

made in the USA. All other copyright questions, including photocopying outside of the USA,
should be referred to the Publisher.
Printed on acid-free paper
Printed in The Netherlands


V

Preface
The first two volumes in the series New Comprehensive Biochemistry appeared in
1981. Volume 1 dealt with membrane structure and Volume 2 with membrane transport. The editors of the last volume (the present editor being one of them) tried to
provide an overview of the state of the art of the research in that field. Most of the
chapters dealt with kinetic approaches aiming to understand the mechanism of the
various types of transport of ions and metabolites across biological membranes.
Although these methods have not lost their significance, the development of molecular biological techniques and their application in this field has given to the area of
membrane transport such a new dimension that the appearance of a volume in the
series New Comprehensive Biochemistry devoted to molecular aspects of membrane
proteins is warranted.
During the last decade hundreds of primary structures of membrane proteins have
been published and each month several new sequences of transport proteins appear in
the data banks. From these sequences global models for the structure of membrane
proteins can be made using several type of algorithms. These models are very useful
for a partial understanding of the structure of these proteins and may help us with
understanding part of the mechanism of action. They d o not, however, provide us
with complete answers of how these pumps, carriers and channels actually function.
The combination of biochemical (site-specific reagents), molecular biological (sitedirected mutagenesis) and genetic approaches of which this volume gives numerous
examples in combination with such biophysical techniques as X-ray analysis and
NMR will eventually lead to a complete elucidation of the mechanism of action of
these transport proteins.
It is clearly impossible to give a comprehensive overview of this rapidly expanding

field. I have chosen a few experts in their field to discuss one (class of) transport
protein(s) in detail. In the first five chapters pumps involved in primary active
transport are discussed. These proteins use direct chemical energy, mostly ATP, to
drive transport. The next three chapters describe carriers which either transport
metabolites passively or by secondary active transport. In the last three chapters
channels are described which allow selective passive transport of particular ions. The
progress in the latter field would be unthinkable without the development of the patch
clamp technique. The combination of this technique with molecular biological
approaches has yielded very detailed information of the structure-function relationship of these channels.
Despite the limitation in the choice of membrane proteins, I hope that this volume
will be useful for teachers, students and investigators in this field. Although only a
limited number of transport proteins is discussed in this volume in detail, the


vi
approaches described here can be applied to other membrane proteins too and may
lead to further progress in our understanding of this fascinating field.
Jan Joep H.H.M. De Pont
Nijmegen, The Netherlands,
January, 1992


vii

List of contributors
Stephen A. Baldwin,
Departments of Biochemistry and Chemistry, and Protein and Molecular Biology,
Royal Free Hospital School of Medicine (University of London), London NW3 2PF,
U.K.
Rebecca M . Brawley,

Department of Pharmacology, Northwestern University Medical School, Chicago, TL
60611, U . S . A .
Chan Fong Chang,
Department of Pharmacology, Northwestern University Medical School, Chicago, I L
6061 I , U.S.A.
Jan Joep H.H.M. De Pont,
Department of Biochemistry, University of Nijmegen, 6.500 H B Nijmegen, The Netherlands.
Rainer Greger,
Physiologisches Institut der Albert-Ludwigs- Universitat, 7800 Freiburg i. Brsg., Germany.
M. Grenson,
Universite' Libre de Bruxelles, Faculte' des Sciences, Dipartement de Biologie Mole'culaire, Laboratoire de Physiologie Cellulaire et de Ge'nPtique des Levures, B-10.50 Bruxelles, Belgium.
Luis M. Gutierrez,
Department of Pharmacology, Northwestern University Medical School, Chicago, IL
60611, U.S.A.
M. Marlene Hosey,
Department of Pharmacology, Northwestern University Medical School, Chicago, I L
60611, U.S.A.
Peter Igarashi,
Department of Medicine, Yale University School of Medicine, New Haven, C T 06.510,
U.S.A.
Peter Leth Jerrgensen,
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen,
2100 Copenhagen OE, Denmark.
J.S. Lolkema,
The BTOSON Research Institute, University of Croningen, 9747 AG Croningen, The
Net her lands.
Anthony Martonosi,
Department of Biochemistry and Molecular Biology, State Universitis of New York

...

Vlll

Health Science Center, Syracuse, N Y 13210, U.S.A.
Cecilia Mundina-Weilenmann,
Department of Pharmacology, Northwestern University Medical School, Chicago, IL
60611, U.S.A.
0. Pongs,
Zentrum fur Molekulure Neurobiologie, 2000 Hamburg 20, Germany.
G.T. Robillard,
The BIOSON Research Institute, University of Groningen, 9747 AG Groningen, The
Netherlands.
Gene A. Scarborough,
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel
Hill, N C 27599, U.S.A.
Tom J.F. Van Uem,
Department of Biochemistry, University of Nijmegen, 6500 H B Nijmegen, The Netherlands.


1x

Contents
P r ~ f u c e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Chapter 1. Nu.K.A TPase. structure and transport mechanism
Peter Leth Jmgensen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. The Na, K-pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Recent review articles on Na. K-ATPase structure and function . . . . . .
2 . Structure of Na, K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Purified membrane-bound and soluble Na.K-ATPase . . . . . . . . . . . . .
2.1.1.
Enzymatic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Electron microscopy and crystal analysis . . . . . . . . . . . . . . .
2.1.3.
Three-dimensional models . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Cytoskeletal associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Proteolytic dissection of membrane-bound Na, K-ATPase . . . . . . . . . .
2.4. Membrane organization of the c1 subunit . . . . . . . . . . . . . . . . . . . . .
2.5. Structure of the fl subunit of Na, K-ATPase . . . . . . . . . . . . . . . . . . .
3 . Nucleotide binding and phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. The nucleotide binding domain in the alp1 unit . . . . . . . . . . . . . . . .
3.1.1.
Comparison with the nucleotide binding sites in adenylate
kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Selective chemical labelling with ATP analogues . . . . . . . . . .
3.2. Conformations of the nucleotide binding area . . . . . . . . . . . . . . . . . .
3.3. The phosphorylation site, high- and low-energy phosphoforms, ElP-E2P
4 . Cation binding and occlusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.1. Capacity for binding and occlusion of Na' or K + ( R b + ) . . . . . . . . . .
4.2. Isolation of the cation occlusion and transport path after tryptic
digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Transport stoichiometry and net charge of N a + and K + complexes
with Na, K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Structural transitions in the protein related to energy transformation and
Na, K-transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Conformation dependent proteolytic cleavage of Na, K-ATPase . . . . . .
5.2. Tryptophan fluorescence and secondary structure changes . . . . . . . . . .
5.3. Cleaved derivatives; cleavage of bond 2 and the regulatory function of
the N-terminus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Effect of C3 cleavage on EIP-E2P transition and cation exchange . . . . .
5.5. Mutagenesis in yeast H-ATPase and Ca-ATPase from sarcoplasmic
reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6. Coupling to ion translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
i
1

2
2
2
3
3
5
6
7
7

10
11

12
12
12
13
13
15
16
17
17

18
18
19
20
20
21
22
23


X

Chapter 2 . Structure and function of gastric H. K.ATPase
Tom J.F. Van Uem and Jan Joep H.H.M. De Pont . . . . . . . . . . . . . . . . . .
1.
2.
3.

27

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tissue and cell distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. The catalytic a subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The fl subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Molecular organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Conformations of H,K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Kinetics of H,K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Overall reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Phosphorylation from ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Characteristics of ATP hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Hydrolysis of p-nitrophenylphosphate . . . . . . . . . . . . . . . . . . . . . . .
4.5. Phosphorylation from inorganic phosphate . . . . . . . . . . . . . . . . . . .
5. Transport by H,K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. The H+-ATP stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
5.2. Ion selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Electrogenicity of ion transport . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Lipid dependency of H,K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Solubilization and reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Inhibitors of H,K-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27
28
28

28
31
34
34
36
36
38
39
40
41
42
42
42
43
44
45
46
49
49

Chapter 3 . The Ca2+ transport ATPases of sarco(endo)plasmic reticulum and
plasma membranes
Anthony Martonosi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57

1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The classification of Ca2'-ATPase isoenzymes . . . . . . . . . . . . . . . . . . . . .
2.1. The Ca2+ transport ATPases of sarco(endo)plasmic reticulum (SERCA)
2.1.1.
SERCAl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
SERCA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3.
SERCA3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.4.
SERCA-type Ca2+-ATPases from non-mammalian cells
(SERCAMED). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. The plasma membrane Ca2+ transport ATPases (PMCA) . . . . . . . . . .
2.2.1.
rPMCA1 and rPMCA2 . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2.
rPMCA3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.
rPMCA4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.4.
hPMCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The deduced amino acid sequences of the fast-twitch and slow-twitch isoforms of
the sarcoplasmic reticulum Ca2'-ATPase . . . . . . . . . . . . . . . . . . . . . . . .
The predicted topology of the Ca2+-ATPases. . . . . . . . . . . . . . . . . . . . . .
4.1. The Ca*'-ATPase of the sarcoplasmic reticulum . . . . . . . . . . . . . . . .
4.1.1.
The cytoplasmic headpiece . . . . . . . . . . . . . . . . . . . . . . . .

51
58
58
58
58
59
59
59
62
62
62
62
62
64
64
65


xi
4.1.1.1. The phosphorylation and nucleotide binding domains . . . . . .
4.1.1.2. The transduction or B domain . . . . . . . . . . . . . . . . . . . . . .
4.1.1.3. The hinge domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1.4. The stalk region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2.
The transmembrane domain . . . . . . . . . . . . . . . . . . . . . . .
4.2. The predicted domains of the plasma membrane Ca2’.- ATPase . . . . . .
5 . Reconstruction of Ca”-ATPase structure by electron microscopy . . . . . . . .
5.1. The vanadate-induced E2-type crystals . . . . . . . . . . . . . . . . . . . . . .
5.1.1.
Image reconstruction in three dimensions from negatively stained

and frozen hydrated crystals . . . . . . . . . . . . . . . . . . . . . . .
5.2. Crystallization of Ca’+-ATPase by Ca” and lanthanides in the E l state
5.3. Crystallization of Ca2 ’ -ATPase in detergent-solubilized sarcoplasmic
reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. X-ray and neutron diffraction analysis of the Ca2+-ATPase of sarcoplasmic
reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Site specific mutagenesis of sarcoplasmic reticulum Ca2’-ATPase . . . . . . . . .
7.1. The search for the Ca2+ binding site . . . . . . . . . . . . . . . . . . . . . . .
7.1.1.
Mutation of amino acids in the stalk sector . . . . . . . . . . . . .
7.1.2.
The probable location of Ca2+ binding sites in the transmembrane
domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Mutations in the putative catalytic site . . . . . . . . . . . . . . . . . . . . . .
7.2. 1.
Mutations around Asp35 1 . . . . . . . . . . . . . . . . . . . . . . . .
7.2.2.
The mutations around Lys5 I S . . . . . . . . . . . . . . . . . . . . . .
7.2.3.
The role of sequences 601-604 in ATP binding and Ca’ ‘
transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.4.
Mutations in the R616-K629 region of the Ca’ -ATPase
(Thr625, Gly626, Asp627) . . . . . . . . . . . . . . . . . . . . . . . .
7.2.5.
Mutations in the 701-707 region . . . . . . . . . . . . . . . . . . . .
7.2.6.
Mutations of Lys712 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.7.
The structure of the ATP binding site . . . . . . . . . . . . . . . . .

7.3. The /3 strand sector . Conformational change mutants . . . . . . . . . . . . .
7.4. The transmembrane segments of the Ca2 ’ -ATPase . . . . . . . . . . . . . .
8 . In situ proteolysis of Ca’&-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1. Hydrolysis of Ca2+-ATPase by trypsin . . . . . . . . . . . . . . . . . . . . . .
8.1.1.
The T I cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.2.
The T2 cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.3.
The cleavage of Ca’ ‘-ATPase by trypsin at the T3 and T4 sites
8.2. The effect of other proteolytic enzymes on the Ca’.‘-ATPase . . . . . . . .
8.2. I .
Chymotrypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.2.
Thermolysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.3.
Staphylococcal V8 protease . . . . . . . . . . . . . . . . . . . . . . . .
8.3. Vanadate-catalyzed photocleavage of the Ca’ ’~ -ATPase . . . . . . . . . . .
9 . Monoclonal and polyclonal anti-ATPase antibodies . . . . . . . . . . . . . . . . . .
9.1. Antibodies reacting with the N- and C-terminal regions of the
Ca2+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2. Distribution of epitopes in the cytoplasmic domain of Ca”-ATPase . . .
9.3. Antibodies reacting with the putative luminal domain of the Ca’- ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Covalent modification of side-chain groups in the Ca’+-ATPase . . . . . . . . .
10.1. Sulfhydryl groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65
67
67
67

68
68
70
70
71
73
73
77
78
78
78
79
79
79
80
80
81
81
81
81
82
83
84
84
84
85
86
87
87
87

87
87
88

89
89
90
91
91


xii
10.1.1.

Identification of cysteine residues that react with N-ethylmaleimide
91
(MalNEt) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.2. The reaction of iodoacetamide and its N-substituted derivatives
92
with the Ca2+-ATPase. . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.2.1. Iodoacetamide (IAA) and 5-(2-acetamidoethyl)aminonaphthalene92
1-sulfonate (IAEDANS) . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.2.2. The reaction of 6-(iodoacetamido)fluorescein (IAF) with the
92
Ca2+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.3. Modification of Ca2+-ATPase with 7-chloro-4-nitrobenzo-2-oxa92
1,3-diazole (NBD-C1) . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1.4. The disulfide of 3’(2’)-O-biotinyl-thioinosine triphosphate (biotin93
yl-S6-1TP2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
93

10.2. Modification of lysine residues . . . . . . . . . . . . . . . . . . . . . . . . . . .
94
10.3. Modification of arginine residues . . . . . . . . . . . . . . . . . . . . . . . . . .
95
10.4. Modification of histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
10.5. Modification of carboxyl groups . . . . . . . . . . . . . . . . . . . . . . . . . .
96
10.5.1. The reaction of Ca2+-ATPasewith dicyclohexylcarbodiimide . .
10.5.2. The reaction of Ca2+-ATPase with N-cyclohexyl-N’-(4-dimethyl97
amino-a-naphthyl) carbodiimide (NCD-4) . . . . . . . . . . . . . .
10.5.3. Reaction of Ca2+-ATPase with the carbodiimide derivative of
97
ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Spatial relationships between functional sites in the sarcoplasmic reticulum
98
Ca2+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
98
1 1.1. Intramolecular distances determined by fluorescence energy transfer . . .
11.1.1. The location of the high-affinity Ca2+ binding site . . . . . . . .
100
101
11.1.2. The ATP binding site . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 1.1.3. The use of IAEDANS as reference point for distance measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
102
11.2. Thermal fluctuations in the structure of the Ca*+-ATPase. . . . . . . . . .
103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
105

Chapter 4 . The Neurospora crassa plasma membrane H + -ATPase
Gene A . Scarborough . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structural features of the H+-ATPase molecule . . . . . . . . . . . . . . . . . . . .
2.1. H+-ATPase conformational changes. . . . . . . . . . . . . . . . . . . . . . . .
2.2. The purified ATPase preparation . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Subunit composition of the H+-ATPase . . . . . . . . . . . . . . . . . . . . .
2.4. The minimum functional unit . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Primary structure of the H+-ATPase . . . . . . . . . . . . . . . . . . . . . . .
2.6. Secondary structure of the H -ATPase . . . . . . . . . . . . . . . . . . . . . .
2.7. Protein chemistry of the H+-ATPase . . . . . . . . . . . . . . . . . . . . . . .
2.8. Chemical state of the H -ATPase cysteines . . . . . . . . . . . . . . . . . . .
2.9. Transmembrane topography of the H+-ATPase . . . . . . . . . . . . . . . .
2.10. A first-generation model for the tertiary structure of the H+-ATPase . .
3. The molecular mechanism of transport . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
+

+

117
117
118
118
119
119
120

121
121
122
122
123
124
129
131


xiii

Chapter 5 . The Enzymes 11 of the phosphoenolpyruvate-dependent carbohydrate transport systems
J.S. Lolkema and G.T. Robillard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 .1. PTS carbohydrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. PTS components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. PTS nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Enzyme I1 structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Sequence homology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Domain function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.
The A domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.
The B domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.3.
The A and B domains of E . coli HIMa" . . . . . . . . . . . . . . . .

2.3.4.
The C domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Domain interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.1.
Association state of E-I1 . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.1.1, E-II'" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.1.2. E-IIGLC
.....................................
2.4.2.
Kinetics of domain interaction . . . . . . . . . . . . . . . . . . . . .
. .
3. Binding studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Equilibrium binding to E-I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Orientation of the binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Kinetics of binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . The coupling between transport and phosphorylation . . . . . . . . . . . . . . . . .
4.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Phosphorylation of free cytoplasmic carbohydrates . . . . . . . . . . . . . .
4.3. Facilitated diffusion catalyzed by E-I1 . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.
Diffusion in uptake studies . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2.
Diffusion in efflux studies . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3.
Regulation of efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Coupling in vectorial phosphorylation . . . . . . . . . . . . . . . . . . . . . . .
5 . Steady-state kinetics of carbohydrate phosphorylation . . . . . . . . . . . . . . . .
5.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. The R . sphaeroides IIF'" model . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3. The E . coli IIMtLmodel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 6 . Mechanisms of active and passive transport in a family of
homologous sugar transporters found in both prokaryotes and eukaryotes
Stephen A . Baldwin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The kinetics of sugar transport in mammalian cells . . . . . . . . . . . . . . . . . .
2.1. Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Specific inhibitors of transport . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Kinetics of transport in the erythrocyte . . . . . . . . . . . . . . . . . . . . . .

135
135
135
135
136
138
138
138
140
140
142
143
143
143
144

144
145
146
147
147
149
149
151
153
153
154
155
155
156
157
158
160
160
161

163
164

169
169
170
170
172
174

xiv
2.3.1.
General properties and methods of investigation . . . . . . . . . .
2.3.2.
Transport asymmetry and the effect of cytoplasmic ATP . . . . .
2.4. Kinetic models for the transport process . . . . . . . . . . . . . . . . . . . . .
2.5. Measurements of individual rate constants for steps in the transport cycle
3. Characterization of the isolated human erythrocyte transporter . . . . . . . . . .
3.1. Purification and kinetic properties of the transporter protein . . . . . . . .
3.2. Molecular properties of the isolated protein . . . . . . . . . . . . . . . . . . .
3.2.1.
Polypeptide composition and glycosylation state . . . . . . . . . .
3.2.2.
Secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Oligomeric state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . The structure of the human erythrocyte glucose transport protein . . . . . . . . .
4.1. Amino acid sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Arrangement in the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2.
Three-dimensional arrangement . . . . . . . . . . . . . . . . . . . . .
4.3. Location of the substrate-binding site(s) . . . . . . . . . . . . . . . . . . . . .
4.3.1.
Insights from proteolytic digestion . . . . . . . . . . . . . . . . . . .
4.3.2.
Photoaffinity labelling with cytochalasin B . . . . . . . . . . . . . .
4.3.3.

Photoaffinity labelling with bis-mannose derivatives . . . . . . . .
4.3.4.
Photoaffinity labelling with forskolin and its derivatives . . . . .
4.3.5.
Photoaffinity labelling with miscellaneous inhibitors . . . . . . . .
5. Conformational changes and the mechanism of transport . . . . . . . . . . . . . .
5.1. Influence of substrates and inhibitors on reactivity towards group-specific
reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Biophysical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Differential susceptibility of conformers to proteolysis . . . . . . . . . . . .
6 . Homologous transporters and their distribution in mammalian tissues . . . . . .
6.1. GLUT-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. GLUT-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. GLUT-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. GLUT-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5. GLUT-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Homologous transporters in other organisms . . . . . . . . . . . . . . . . . . . . . .
7.1. Fungal transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Protozoan transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. The transporters of photosynthetic organisms . . . . . . . . . . . . . . . . . .
7.4. Bacterial transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The galactose, arabinose and xylose transporters of E . coli . . .
7.4.1.
7.4.1.1. The D-xylose/Hf transporter . . . . . . . . . . . . . . . . . . . . . .
7.4.1.2. The L-arabinose/H transporter . . . . . . . . . . . . . . . . . . . .
7.4.1.3. The D-galactose/H + transporter . . . . . . . . . . . . . . . . . . . . .
7.4.2.
The citrate and tetracycline transporters of E . coli . . . . . . . . .
7.4.3.
The lactose transporter of E . coli . . . . . . . . . . . . . . . . . . . .

8 . Clues to the mechanism of transport from comparison of the homologous
transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
+

174
176
177
179
182
182
184
184
184
185
185
185
186
186
189
189
189
189
190
191
191
192
192
194

195
196
197
198
199
199
200
200
200
201
201
202
202
202
202
202
203
207
208
210
21 1


xv
Chapter 7. Amino acid transporters in yeast: structure. .function and regulation
M.Grenson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Physiological background: assimilation of exogenous nitrogen compounds used
as a source of nitrogen or as building blocks . . . . . . . . . . . . . . . . . . . . . .
3 . General characteristics of amino acid transporters in Saccharomyces cerevisiae .

3.1. Accumulation of amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Multiplicity and specificity of amino acid transporters in Saccharomyces
cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Functional specialization of amino acid transporters . . . . . . . . . . . . .
3.4. Irreversibility of amino acid accumulation . . . . . . . . . . . . . . . . . . . .
3.5. Role of the vacuole in amino acid retention . . . . . . . . . . . . . . . . . . .
3.6. Efflux of amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Identifying transport systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Isolating mutants affected in uptake systems . . . . . . . . . . . . . . . . . .
4.2. An example of transporter identification in a complex case: the three
GABA transport systems of Saccharomyces cerevisiae . . . . . . . . . . . . .
5 . Structure and evolution of amino acid transporters . . . . . . . . . . . . . . . . . .
5.1. Molecular cloning and nucleotide sequencing of amino acid permease
genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. A family of amino acid transporters with amino acid sequence homologies
6. Regulation of amino acid transport . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Regulation of permease activity . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Regulation of permease synthesis . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1.
Case of the NCR-insensitive amino acid permeases . . . . . . . .
6.2.2.
Case of the NCR-sensitive amino acid permeases . . . . . . . . .
6.2.2.1. Constitutive expression of permease genes . . . . . . . . . . . . . .
6.2.2.2. Inducible permeases . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Nitrogen-catabolite repression (NCR) and nitrogen-catabolite inactivation
(NCI): two superimposed regulatory mechanisms affecting uptake systems
for nitrogenous compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.1.
Regulation of amino acid permease activity as a function of
nitrogen availability . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.3.1.1. Nitrogen-catabolite inactivation (NCI): negative control of GAP1
activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.1.2. Positive control of GAP1 activity . . . . . . . . . . . . . . . . . . . .
6.3.1.3. How is GAP1 activity regulated? . . . . . . . . . . . . . . . . . . . .
Nitrogen-catabolite repression (NCR) . . . . . . . . . . . . . . . . .
6.3.2.
6.3.2.1. N C R affects permease gene transcription o r transcript accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.2.2. Glutamine as an effector of NCR . . . . . . . . . . . . . . . . . . .
6.3.2.3. The UREZIGDHCR gene product as a negative regulatory protein
which participates in the repression of permease synthesis . . . .
6.3.2.4. The GLN3 gene product as a possible target for the URE2I
GDHCR gene product . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.2.5. Double regulation of the ammonia-sensitive permeases . . . . . .
I . The APFl gene product. a common factor of unknown function which increases
the activity of amino acid permeases . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Summary and prospects . . . . . . . . . . . . . . . . . .

1.
2.

219
219
220
222
222
222
223
223
224
225

225
226
226
221
221
23 1
232
232
234
234
234
234
235

231
238
238
239
239
240
240
240
240
240
241
241
241


XVI

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243

Chapter 8 . Structure and function of plasma membrane Na+lHt exchangers
Peter Igarashi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

247

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. The N a t / H + exchanger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Functional heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Biochemical properties of N a + / H + exchangers . . . . . . . . . . . . . . . . . . . . .
2.1. ‘Group-specific’ modification . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
Imidazolium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Carboxyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3.
Sulfhydryl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.4.
Amino . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.5.
Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Identification and characterization of candidate transport protein(s) . . .
2.2.1.
Covalent labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.2.
Affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.
Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Molecular cloning of N a + / H + exchangers . . . . . . . . . . . . . . . . . . . . . . . .
3.1. cDNA cloning and primary structure . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Human N a + / H + exchanger cDNA . . . . . . . . . . . . . . . . . .
3.1.2. Other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Tissue and membrane localization . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Isofoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Genomic cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Summary and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

247
247
248
249
249
250
251
252
253
254
255
255
257
258
260

260
261
263
265
267
268
269
270

Chapter 9 . Cl- -channels
Rainer Greger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

273

1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Different types of CI-.channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. C1-. channels in the nervous system . . . . . . . . . . . . . . . . . . . . . . . .
2.2. C1-. channels of muscle and electric organ . . . . . . . . . . . . . . . . . . . .
2.3. C1-. channels in apolar non-excitable cells . . . . . . . . . . . . . . . . . . . .
2.4. The problem of detecting small C1-. channels . . . . . . . . . . . . . . . . . .
2.5. Epithelial C1-. channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The structure and molecular basis of CI-. channels . . . . . . . . . . . . . . . . . .
3.1. The GABAA-receptor and glycine-receptor channels . . . . . . . . . . . . . .

3.2. The Torpedo marmorata C1-. channel . . . . . . . . . . . . . . . . . . . . . . .
3.3. Muscle Cl-.channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Epithelial C1-. channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmacological modulation of CI-.channels . . . . . . . . . . . . . . . . . . . . . .
4.1. Pharmacological modulation of GABAA-receptor and glycine-receptor
channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Inhibition of epithelial Cl-.channels . . . . . . . . . . . . . . . . . . . . . . . .

273
274
275
276
276
277
278
280
281
281
282
282
283
283
284


xvii
Regulation of epithelial CI.. channels . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. The CI-. channel defect in cystic fibrosis . . . . . . . . . . . . . . . . . . . . .
5.2. Mechanisms of CI-. channel activation in epithelia . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

287
288
290
291

Chapter 10. Voltage-gated K+ channels
0. Pongs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297

5.

1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure and biophysical properties of cloned voltage-gated K+channels . . . .
2.1. K' channels of the Shokrr family . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. K' channels of the MBK/RCK/HBK family . . . . . . . . . . . . . . . . . .
2.3. K + channels of Shaker relatives . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Pharmacology of K + channels . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Structure of K' channel genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Genes in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Vertebrate genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . The basis of K + channel diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Properties of homo- and heteromultimers . . . . . . . . . . . . . . . . . . . .
4.2. Functional domains in K + channels . . . . . . . . . . . . . . . . . . . . . . . .
5 . General structural implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297
298
298
301
302
302
306
306
307
307
307
308
309
311

Chapter I 1 . Structure and regulation of voltage-dependent L-type calcium
channels
M . Marlene Hosey. Rebecca M . Brawley. Chan Fong Chang. Luis M .
Gutierrez and Cecilia Mundina- Weilenmann . . . . . . . . . . . . . . . . . . . . . . .

315

1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Subtypes of Ca2+ channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Functions of L-type Cali channels . . . . . . . . . . . . . . . . . . . . . . . .
L-type Ca2+ channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Biochemical and molecular characterization . . . . . . . . . . . . . . . . . . .
2.2.1.
Isolation and purification of the multisubunit dihydropyridinesensitive Ca2+ channels from skeletal muscle . . . . . . . . . . . .
2.2.2.
Identification and purification of L-type channel proteins from
other cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.
DNA cloning and expression of channel proteins . . . . . . . . .
2.2.3.1. Isoforms of the a , subunit . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.2. Cloning of DNAs for other putative channel subunits . . . . . .
2.2.4.
Roles of subunits of L-type Ca2+ channels . . . . . . . . . . . . .
2.2.5.
Reconstitution of Ca2+ channels . . . . . . . . . . . . . . . . . . . .
2.3. Regulation of Ca2+ channels by protein phosphorylation and G-proteins
2.3.1.
Phosphorylation by CAMP-dependent protein kinase . . . . . . .
2.3.2.
Regulation of L-type channels by PKC and other protein kinases
2.3.3.
Regulation of L-type channels by phosphoprotein phosphatases

315
315
317
318
318
319
320

321
322
322
323
324
325
326
327
329
330


xviii
2.3.4.
Regulation of Ca2+channelsby G-proteins . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..
..

331
332

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

337

De Pont. Molecular Aspects of’ Trunsport pro lei ti^
0 1992 Elsevier Science Publishers B.V. All rights reserved

1
CHAPTER 1

Na,K-ATPase, structure and transport
mechanism
PETER LETH JORGENSEN
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen,
2100 Copenhugen OE, Denmark

1. Introduction
1.1. The Na,K-pump

The Na,K-pump is ubiquitous and located at the surface membrane of most animal
cells. Primary active Na,K-pumping is a key process for the active uptake of nutrients, salts and water and for the regulation of fluid and electrolyte homeostasis in
mammals. The pump maintains electrochemical gradients for Na ’-(ApNa) for utilization in carrier mediated secondary active transport processes in kidney, intestine,
lung and other epithelia. In coupling the hydrolysis of ATP to active transport of
3Na+ out and 2 K+ into the cell, the pump is electrogenic and maintains ion gradients required for regulation of cell volume and the pump works as a battery for the
electrical activity of excitable cells.
The Na,K-pump was first purified from the outer medulla of mammalian kidney
[ 1,2] and the protein was characterized in membrane-bound form and in detergent
solution [3,4]. This is a preparation of choice in studies of protein structure and
membrane organization [S], conformational transitions coupled to ion translocation
[6] and identification of sites for binding nucleotides and cations [7]. By incubation in
vanadate medium, the proteins of the purified membrane-bound pump protein can be
organized in crystalline arrays with the afl unit as the minimum asymmetric unit [5,8].
Low resolution models of the overall structure of the Na,K-pump molecule can be
constructed on the basis of diffraction analysis of p l and p12 crystals [9-111. This

knowledge of pump structure and function has been important for understanding the
physiological function and regulation of the Na,K-pump in kidney [12,13].
Application of recombinant DNA techniques led to the primary structure of the x
subunit [ 14,151 and p subunit [ 15,161 of the Na,K-pump in mammalian kidney and a
number of tissues and species (for [ 171 and [ 181). The unitary concept that the Na,K-


2
pump was essentially the same protein in all tissues and cells has been abandoned as
three structurally distinct a subunit isoforms were identified in several species [17,19].
Three j subunit isoforms have now also been disclosed and extensive studies of tissue
specific and developmental expression of the genes encoding the isoforms and the
hormonal regulation of their expression are now being reported. In functional and
regulatory terms, the significance of the expression of the different combinations of
the a l , a2, and a3 or j 1 , j 2 , and j 3 subunits in brain, skeletal muscle, heart and other
tissues remains obscure, mainly because so little is known about the Na,K-transport
and enzymatic properties of isozymes other than the renal (alpl) Na,K-pump.
The a1j l isozyme of Na,K-ATPase remains the ‘household’ pump that is expressed
in kidney, other epithelia and most other cells. This chapter is focused on the
structural organization of this renal Na,K-pump and the molecular mechanisms
behind the transformation of chemical energy to movement of N a t and K + across
the membrane. Particular emphasis is placed on the organization of the proteins in
the membrane, their interaction with cytoskeletal components, and identificition of
protein segments that are engaged in binding of nucleotides or cations and in
conformational changes in the protein that bring about a reorientation of the cation
binding sites.
1.2. Recent review articles on Na,K-ATPase structure and function

The expansion of our knowledge of the structure and function of Na,K-ATPase is
reflected in a rapid succession of reviews on Na,K-ATPase genes and regulation of

expression [17], subunit assembly and functional maturation [20], the isozymes of
Na,K-ATPase [18], and the stability of c1 subunit isoforms during evolution [21],
physiological aspects and regulation of Na,K-ATPase [22], reconstitution and cation exchange [23], chemical modification [24], and occlusion of cations [25]. Other
valuable sources are the review articles [26] and recent developments [27] reported at
the International Na,K-pump Conference in September 1990.

2. Structure of Na,K-ATPase
2.1. Purified membrane-bound and soluble Na,K-ATPase
The procedure for purification of Na,K-ATPase in membrane-bound form from the
outer renal medulla of mammalian kidney offers the opportunity of exploring the
structure of the Na,K-pump proteins in their native membrane environment. The
protein remains embedded in the membrane bilayer throughout the purification
procedure thus maintaining the asymmetric orientation of the protein in the basolateral membrane of the kidney cell in the purified preparation. This preparation has
been particularly useful in studies of ultrastructure, protein conformation and for


3

identification of sites for binding of ATP and cations [5-71. A further advantage is
that the preparation from outer medulla contains only the alp1 isozyme of Na,KATPase, while most other preparations consist of two or three a subunit and 8subunit isoforms.
The membrane-bound preparation from kidney is easily solubilized in non-ionic
detergent and analytical ultracentrifugation shows that the preparation consists
predominantly (80-85%) of soluble aB units with M , 143000 [28]. The soluble ap
unit maintains full Na,K-ATPase activity, and can undergo the cation or nucleotide
induced conformational transitions that are observed in the membrane-bound preparation. A cavity for occlusion of 2Kf or 3Na+ ions can be demonstrated within the
structure of the soluble aP unit [29], as an indication that the cation pathway is
organized in a pore through the afi unit rather than in the interphase between subunits
in an oligomer.
2.1.1. Enzymatic properties
In an ideal pure preparation of Na,K-ATPase from outer renal medulla, the a1

subunit forms 65-70% of the total protein and the molar ratio of a to fl is 1 : I ,
corresponding to a mass ratio of about 3 : 1 [1,5]. Functionally the preparation
should be fully active in the sense that each a/3 unit binds ATP, Pi, cations and the
inhibitors vanadate and ouabain. The molecular activity should be close to a maximum value of 7 000-8 000 Pi/min. The highest reported binding capacities for ATP
and phosphate are in the rang6 5-6 nmol/mg protein and close to one ligand per c$
unit [29], when fractions with maximum specific activities of Na,K-ATPase [4050 pmol P,/min mg protein) are selected for assay.
2.1.2. Electron microscopy and crystal analysis
The purified membrane bound Na,K-ATPase consists of disc-shaped membrane
fragments, 1 000-3 000 in diameter, with no tendency for vesicle formation. The
densely packed protein particles with diameters of 30-50 represent ap units that
can be visualized by negative staining with phosphotungstic acid or uranyl acetate.
They are arranged in irregular clusters or strands and appear to be free to move in
the plane of the membrane without formation of well defined oligomeric structures
[30]. From negatively stained images similar to those shown in Fig. 1, the average
density of protein in the membrane is estimated to be 12000 ap units/pm2. This
corresponds to a concentration of a subunit in the lipid bilayer of about 7mM or
0.5-1 g protein/ml of lipid phase. These are conditions for supersaturation and formation of crystalline arrays of the protein units in the membrane fragments is
rapidly induced in the presence of vanadate that stabilize the protein in a state
similar to the E2P conformation [ 3 11. In Na,K-ATPase, the predominant crystal
form, shown in Fig. 2a, has the two-sided plane group symmetry, p l , and contains
one protomeric aP unit per unit cell. Crystals with two-sided plane group symmetry,
p21, with two UP units occupying one unit cell, are transient and less frequent

A

A


4

5

a

b

Fig. 2. Crystalline arrays of Na,K-ATPase in the membrane with (a) a protomeric ab unit as minimum
asymmetric unit in a p l crystal or (b) with an oligomeric (a& unit in the unit cell of a p21 crystal. The p l
crystal was formed after incubation of purified membrane-bound Na,K-ATPase in 0.25 mM sodium
monovanadate, 1 mM MgC12 at 4°C. For formation of the p21 crystal the purified membrane-bound
Na,K-ATPase was incubated in 12.5mM phosphate, 1 mM MgC12 and lOmM Tris-HCI, pH 7.5 at 4°C.
The membranes were negatively stained with uranyl acetate and micrographs were obtained at 235000 x
magnification. Images suitable for further analysis were densitometered at 20-pm intervals. Projection
maps were calculated using the Fourier transform amplitudes and phases collected a t the reciprocal lattice
points. The protein-rich regions are drawn with unbroken contour lines, while negative stain regions have
dashed lines. In the reconstructed images 1 mm corresponds to 2.8
The unit cell dimensions are in a:
~ - 5 3 A , h = 5 1 A , y = 1 2 0 " ; a n d i n b : a = 1 3 5 A , h = 4 4 A , y = 1 0 I 0 . From[33].

A.

[32,33], Fig. 2b. The appearance of two crystal forms shows that the protein in the
membrane exists in equilibrium between the protomeric ap unit and oligomeric (alj)2
forms. The high rate of crystal formation of the protein in vanadate solution shows
that transition to the E2 form reduces the difference in free energy required for self
association of the protein. This vanadate-method for crystallization has been very
reproducible [34-361 and it also leads to crystalline arrays of Ca-ATPase in sarcoplasmic reticulum [37] and H,K-ATPase from stomach mucosa [38].

2.1.3. Three-dimensional models

Low resolution models (20-30 A) based on diffraction analysis of membrane crystals
of Na,K-ATPase [34,35,39] and Ca-ATPase [40,41] show that the cytoplasmic protrusions of the proteins are remarkably similar. A notable difference is a 1&20A
Fig. 1. Negative staining by phosphotungstic acid of Na,K-ATPase purified in membrane-bound form.
The membrane surfaces are covered by particles arranged in clusters between smooth areas. From [2]
procedure as described by Deguchi et al. [30].


protrusion on the extracellular surface of the model for Na,K-ATPase while the CaATPase model has a smooth extracytoplasmic surface.
The limitation of the resolution of these reconstructions is the internal order of the
Na,K-ATPase crystals. At the given resolution of 20-25
some interactions and
basic structure characteristics can be resolved, but there is no assignment of structural
detail. Higher resolution has been obtained in studies of Ca-ATPase from sarcoplasmic reticulum in lamellar arrays consisting of sheets of protein arrays separated by
lipid layers that are prepared from soluble Ca-ATPase in non-ionic detergent [42,43].
Thin three-dimensional crystals were grown by adding purified Ca-ATPase to
appropriate mixtures of detergent, lipid and calcium [44]. They are rapidly frozen
and maintained in frozen-hydrated state during electron microscopy. Electron
diffraction extends to 4 A and images contain phase data to 6 A resolution. Based
on these projections and the previously determined low-resolution structure of CaATPase a packing diagram for the three-dimensional crystals is presented. A model
with a specific arrangement for ten transmembrane a-helices is proposed [45].

A

2.2. Cytoskeletal associations
In the polarized tubule cells of mammalian kidney, the specific associations of
Na,K-ATPase with cytoskeletal components and the cellkell contacts appear to be
important for the induction of polarity of the ap unit between luminal and basolatera1 membranes. The epithelial cell adhesion molecule (CAM) uvomorulin (cadherin) functions as an inducer of cell polarity for the constitutively expressed alp1 units
through cytoplasmic linkage to the membrane cytoskeleton. Loss of polarity with
incorrect localization of Na,K-ATPase to apical membranes has been associated
with a number of diseases including polycystic kidney disease [46,47]. One link to

the cytoskeleton is a high affinity binding site for ankyrin (& = lop8) that has been
demonstrated in the purified renal Na,K-ATPase [48]. A fraction of the alp1 units
seems to associated in Na,K-ATPase-ankyrin-fodrin complexes with similarity to
the capnophorin-ankyrin-spectrin complexes in the cytoskeleton of the human erythrocyte. Induction of cellkell contact alters the properties and distribution of these
proteins. Before contact between the epithelial cells, the ankyrin-fodrin tetramers
form complexes with the membrane proteins, either Na,K-ATPase or uvomorulin.
On cellkell contact, uvomorulin seems to mediate redistribution so that the Na,KATPase-ankyrin-fodrin complexes accumulate at the sites of cellkcell contact
[49,50]. It is proposed that the cellkell contacts via uvomorulin induce the specific
distribution at the cell surface of Na,K-ATPase during development of the polarized
epithelial cells. Once polarity has been established, the proteins are replaced by
targeting to the appropriate membrane from the Golgi complex.
Neuron-glial adhesion in nerve cell cultures is mediated by the 82 subunit AMOG
(adhesion molecule on glia) in the a2p2 isozyme of Na,K-ATPase [51]. Antibodies to
the 82 subunit dissociate cellkcell associations and also increase the rate of active