Director,Office of Research Advocacy OHSU
Senior Scientist,Divisions of Reproductive Sciences and
Neuroscience ONPRC
Professor,Departments of Pharmacology and Physiology,Cell and
Developmental Biology,and Obstetrics and Gynecology OHSU
Beaverton,Oregon
PART I
Assembly
CHAPTER 1
Assembly of Ion Channels
ZuFang Sheng and Carol Deutsch
Department of Physiology
University of Pennsylvania
Philadelphia
Pennsylvania
USA
I.Introduction
II.Strategies and Methods
A.Identification of Putative Regions Involved in Intersubunit
Interactions
B.Characterization of Intersubunit Interactions
C.Determination of Subunit Stoichiometry and History During
Assembly
References
I.Introduction
Most ion channels are multisubunit conglomerates.Because synthesis
and assembly
of many different types of pore-forming subunits occur in a single
cell,how
do the right subunits find each other to give the correct
stoichiometry and avoid
scrambling to channel homogeneity?This problem is even more
striking if we
consider the vast number of nonchannel transmembrane proteins made
simultaneously
in a cell.Assembly is a multistep process that requires specific
intersubunit
recognition events.Each of these steps may include intermediate
folded conformations
of subunits andor intermediate subunit stoichiometries.Such
possibilities
have not been explored for most types of ion channels,including Kt
channels,nor
is it known which regions of the subunits actually interact during
each assembly
step.
In some cases,the NH2-terminal domains of ion channels can function
as
specific recognition motifs between subunitsBabila et al.,1994;Li
et al.,1992;
Shen et al.,1993;Verrall and Hall,1992;see also Xu and Li,1998,this
volume,but
it is not clear that such elements contribute to stabilization of
the mature multimeric
protein or whether additional subunit–subunit interactions
between
transmembrane segments provide the energy to shift the equilibrium
in a
lipid bilayer toward multimerization and the final,mature channel
that functions
in the plasma membrane.Most voltage-gated Kt channels are
homotetrameric
membrane proteins,each subunit containing six putative
transmembrane segments,
S1–S6.It is not clear what holds the tetramer together;intersubunit
covalent
linkages do not appear to be responsibleBoland et al.,1994.In
these
channels the cytoplasmic NH2 terminus contains a recognition
domain,T1
“first tetramerization”,that tetramerizes in vitro and confers
subfamily specificity
Li et al.,1992;Shen and Pfaffinger,1995;Shen et al.,1993;Xu et
al.,1995.
However,in the native channel there are also intramembrane
associationIMA
sites in the central core of voltage-gated Kt channels that provide
sufficient
recognition and stabilization interactions for channel assembly,and
disruption
of one or more of these interactions may suppress channel
formationSheng et al.,
1997;Tu et al.,1996.The relative contributions of different domain
interactions
e.g.,T1 and IMAmay vary from channel isoform to isoform.What are
these
T1 and IMA domains in the native full-length Kt channel,and what
are their
relative contributions to channel formation?
Identification of the recognition and stabilization motifs in the
primary sequence
of channel proteins is a good beginning to understanding channel
assembly;
however,it still leaves many questions unanswered.How specific are
these intersubunit
interactions?How strong are they? At which stage in assembly are
subunits
integrated into the membrane?What are the spatial and temporal
events involved
in channel assembly?What is the subunit stoichiometry of the
channel?What is the
history of the subunits during assembly?Is recruitment of subunits
a random
event?What is the nature of the subunit pool?Where is it
located?When are
subunits recruited into multimeric channels,and where?We can
address these
issues both biochemically and biophysically,as described in the
next section,
using a variety of in vitro translation systems and in vivo
expression systems.
The in vitro translation systems include rabbit reticulocyte
lysateRRLand
wheat germ agglutininWGAsystems,which contain cellular components
necessary
for protein synthesistRNA,ribosomes,amino acids,and
initiation,elongation,
and termination factorsand are capable of a variety of
posttranslational
processing activitiesacetylation,isoprenylation,proteolysis,and
some phosphorylation
activity.Signal peptide cleavage and core glycosylation can be
reconstituted
and studied by adding canine pancreatic microsomal membranes to
the
translation reaction.These systems permit studies,for example,of
transcriptional
and translational control,association of proteins,and their
membrane integration.
However,the translation efficiency of high molecular weight
proteins100,000is
relatively poor,and it is not clear that all aspects of in vivo
processing have been
reconstituted.Thus,caution must be used in extrapolating findings
with the in vitro
system to in vivo events.
The in vivo expression system most used for study of channel
function and
assembly has been Xenopus oocytesRudy and Iverson,1992.Mammalian
cells
are also used frequently and involve DNA transfection
techniquesRudy and
Iverson,1992.Oocytes typically require injection of channel
mRNAtypically
50 nloocyte;0.1–100 ng mRNAoocyte.This system is an intact
cell system that
expresses at high levels for both electrophysiological and
biochemical measurements,
which can be done simultaneously in parallel samples.Both the
oocyte and
a mammalian T-cell expression system are described later,as well as
the methods
used to study channel protein synthesis,integration into
membranes,and
oligomerization.
Broadly defined,assembly also involves
trafficking,posttranslational modification,
and localization of channel proteins in specific subcellular
compartments,as
well as the aforementioned processes of recognition and
associationoligomerization.
This chapter,however,focuses only on strategies and methods that
can be
used1to identify regions of a protein that are potentially
involved in intersubunit
interactions during assembly of the pore-forming unit of ion
channels,2to
determine the strength,kinetics,spatial,and temporal
characteristics of the intersubunit
interactions,and3to determine the subunit stoichiometry and
history of
subunits during assembly.For some cases we illustrate the
approaches by describing
experiments in our laboratory involving a voltage-gated Kt
channel,Kvl.3.
However,these strategies and methods can be,and have been,used for
other
multimeric channels.
II.Strategies and Methods
The strategies used to address the issues just stated entail either
direct or indirect
determinations of various aspects of subunit association.The former
category
includes primarily biochemical approaches;the latter makes use of
functional
readouts.These strategies are protein based,yet each can have
additional strategies
at the DNA level.For example,strategies that entail constructing
genes that link
multiple channel domains in tandem,swapping channel domains to
create chimeras,
andor deleting or mutating domains can be combined with the
protein
assays to elucidate mechanisms of channel assembly.
A.Identification of Putative Regions Involved in Intersubunit
Interactions
Intersubunit association can be assessed by direct and indirect
methods as
described in the following subsections.To discover which regions of
the channel
interact across subunit boundaries,physical association between
channel subunits
or between peptide fragments of a channel and the full-length
channel protein must
be demonstrated.This can be done directly by1immunoprecipitation
of one
member of a complex by antibody against the other
member,2cross-linking
interacting proteins using bifunctional reagents,or3binding
assays of interacting
peptides.Such binding assays have been employed to show that Kt
channel
subunits,or parts of these subunits,multimerize both in vitro and
in vivoBabila
et al.,1994;Li et al.,1992;Shen and Pfaffinger,1995;Shen et
al.,1993.But these
studies have been concerned primarily with cytoplasmic NH2-terminal
interactions.
We describe one of these methods used in our
laboratory,namely,immunoprecipitation.
One important caveat concerning the association of peptide
fragments of a channel with the channel protein is that it is not
clear that such
association faithfully reflects native associations between
full-length subunits
in situ.For instance,constraints imposed on a segment of the
channel in the
context of the full-length folded protein may lead to different
interactions with
another subunit compared with the isolated truncated channel
peptide fragment.
Therefore,for a transmembrane segment,it is ultimately important to
determine
not only whether these interactions occur in the native protein but
also the
topology and orientation of the peptide fragment.
1.Immunoprecipitation
This method requires the use of antibodiesantiserato a protein or
a peptide
construct.If the antibodies to native epitopes are not sufficiently
good,an epitope
tag may be used;c-mycMEQKLI-SEEDLEvans et al.,1985is excellent
for this
purpose.Such nonnative epitopes,however,should be inserted into a
primary
sequence at a nonperturbing distance15 amino acidsfrom
putative topogenic
determinants.The first step in this approach involves making the
appropriate
plasmid DNA either for use in transfections for subsequent in vivo
expression,or
for in vitro transcription to produce mRNA for subsequent use in
either in vivo or
in vitro experiments.Standard methods of restriction enzyme
analysis,agarose gel
electrophoresis,and bacterial transformation are used for these
studies.Plasmid
DNA are purified using Qiagen columnsValencia,CA,and capped mRNA
is
synthesized in vitro from linearized templates using Sp6 or T7 RNA
polymerase
Promega,Madison,WI.
For in vitro immunoprecipitation experiments,proteins are
translated in vitro
with [35S]methionine2 ml25 ml translation mixture; 10 mCiml
DupontNEN
Research Products,Boston,MAin RRLcommercial preparations are
available
from Promega,and from MBI Fermentas,Amherst,NY;laboratory
preparations
can be made according to Jackson and Hunt,1983;Walter and
Blobel,1983in the
presence1.8 ml membrane suspension25 ml translation mixtureor
absence of
canine pancreatic microsomal membranesPromega or MBI
Fermentas,according
to the Promega Protocol and Application Guide.Two proteins that are
proposed
to interact are then cotranslated.Relative mRNA concentrations
should be
determined from the efficiencies of each construct to yield protein
ratios that are
desired.To maximize coimmunoprecipitation,microsomal membranes
should be
used in limiting concentration compared with the total mRNA
concentration.The
translation reaction can be visualized and quantitated using
SDS–PAGE and
phosphor imaging.
To perform immunoprecipitation from an in vitro translation
systemRRL,
microsomal membranes,1–5 ml of cell-free translation products is
mixed in 400 ml
of buffer A [0.1 M NaCl,0.1 M TrispH 8.0,10 mM EDTA,and
1%vv
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