From the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637
With advances in recombinant DNA, structural, and
electrophysiological techniques, much progress has
been made in understanding the structure and, in particular, the function of ion channels. Less progress has
been made in resolving the cell biological events that
guide the assembly and trafficking of these proteins
and that have a major impact on both structure and
function. The assembly of an ion channel refers to the
processes that transform newly synthesized, unfolded
channel subunits into functional ion channels. The
precise mechanisms by which any protein folds and assembles are unknown and the question of how proteins
fold remains a major challenge in biology, attracting
widespread attention (e.g., Brooks et al., 1998 The assembly of ion channels shares many features
with other proteins produced in the secretory pathway.
Certain viral membrane proteins, such as influenza hemagglutinin (HA), have been particularly well studied
(Hammond and Helenius, 1995 While the assembly of ion channels in many respects
is similar to that of other proteins produced in the
secretory pathway, important differences are beginning
to emerge (see Green and Millar, 1995 Another feature of ion channels that distinguishes
them from many other secretory pathway proteins is
that their production, degradation, and subcellular location are under tight regulatory control. In addition
to the subunits that form the functional ion channel
unit, there are a host of "auxiliary" subunits (Gurnett and Campbell, 1996 Difficulties in Assaying Assembly
A variety of techniques are used to assay ion channel assembly (for recent reviews see Sheng and Deutsch,
1998 Ion channel assembly is a dynamic process. To establish the precursor-product sequence of events that occurs during assembly, one must isolate and identify intermediates and follow them as they form and disappear during assembly. The only way to achieve this
objective is through kinetic measurements. Small soluble proteins can be studied at high concentrations in a
test tube, which allows precise biophysical measurements of protein folding and unfolding. Unfortunately,
large biological ion channels do not assemble in a test
tube. Most ion channels cannot even be assembled using in vitro translation methods (for an exception, see
Rosenberg and East, 1992 Another problem inherent in assays of assembly is
the need to solubilize cells to isolate assembly intermediates formed intracellularly. Detergents used to solubilize membranes may cause the dissociation of subunits
in partially assembled complexes even though the fully assembled ion channel is stable in the detergent. The
associations between chaperone proteins and channel
subunits also can be dissociated by detergent (Ou et al.,
1993 Models of AChR Assembly
The muscle-type AChR continues to be the best characterized ion channel in terms of its assembly, though
much is also known about K+ channel and CFTR assembly. AChRs are composed of four distinct, yet homologous subunits,
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References
; Dobson
and Ellis, 1998
). Because single ion channels control
the flow of ~107 ions/s, the malfunction or improper
targeting of even a few channels can be disastrous for a
cell. To avoid this, ion channel assembly and targeting
must occur with almost perfect fidelity. A good example of the consequences of channel misassembly is the
F508 mutation of the cystic fibrosis transmembrane
regulator (CFTR), the cause of most cases of cystic fibrosis. The disease results from misfolding of the protein, which prevents CFTR delivery to the cell surface
(Cheng et al., 1990
).
). It has been established that specialized mechanisms exist to assist in the
folding and assembly of proteins produced in the secretory pathway. The same mechanisms appear to apply to
ion channels. To start, mRNAs are selectively targeted
to the endoplasmic reticulum (ER) membrane where
assembly begins. The initial assembly events are cotranslational. These events include: (a) membrane insertion
of subunits, (b) a set of different processing events such
as attachment of the core N-linked glycan and signal sequence cleavage, and (c) initial rapid folding. Because
these events are cotranslational, they proceed from the
NH2 terminus to the COOH terminus and establish a
vectoral order to the assembly. The rapid cotranslational events are followed by slower folding reactions
where different domains can interact and other types
of processing occur, such as disulfide bond formation
and proline isomerization. For the homotrimeric viral
glycoproteins, such as HA and vesicular stomatitis virus glycoprotein, the subunits undergo a series of slow folding reactions and disulfide rearrangements (Braakman
et al., 1992
; de Silva et al., 1993
). The slow posttranslational folding and processing precede and are a prerequisite for subunit oligomerization. Posttranslational folding, processing, and ultimately oligomerization of
virtually all secretory pathway proteins occur in the ER,
which provides "quality control" by identifying and degrading any misassembled proteins (Hurtley and Helenius, 1989
; Helenius et al., 1992
; Kopito, 1997
).
). One reason
for these differences is that ion channels are larger and
more complex than most proteins. Almost all ion channels are heteromeric and appear to require a set subunit composition, stoichiometry, and the correct positioning of each subunit within the oligomer for proper
function. Another complication with respect to assembly is that subunits are typically polytypic, with anywhere from 2 (e.g., inward-rectifying K+ channels) to
24 (e.g., voltage-gated Na+ channels) membrane spanning domains. As a consequence of their complex
structure, ion channel assembly is slower and less efficient than that of many other membrane proteins. For
nicotinic acetylcholine receptors (AChRs; Merlie and
Lindstrom, 1983
), voltage-gated Na+ channels (Schmidt
and Catterall, 1986
), and the cystic fibrosis transmembrane regulator (Ward and Kopito, 1994
), assembly occurs in 2-3 h and only 20-30% of synthesized subunits
are assembled.
; Sheng and Wyszynski, 1997
; Colledge and Froehner, 1998
; Trimmer, 1998b
) that help to
regulate the expression, targeting, and stability of ion
channels. While some of these subunits assemble with
ion channels in the ER (Nagaya and Papazian, 1997
),
others assemble in the Golgi stacks (Schmidt and Catterall, 1986
) and at the plasma membrane (Froehner
et al., 1990
; Phillips et al., 1991
). Thus, many ion channels differ from other secretory pathway proteins in
that they continue to oligomerize with auxiliary subunits after release from the ER. The addition of these
subunits at sites closer to or at the plasma membrane is
likely to be important for the regulation of ion channel function.
; Trimmer, 1998a
; Xu and Li, 1998
), but few directly assay assembly. The most common methods measure the expression of the fully assembled ion channel either by a functional assay such as electrophysiological
or flux measurements, or by tagging the channel by
means such as metabolic labeling. Subunit regions involved in assembly are inferred by alterations using recombinant DNA methods and measuring how these changes affect expression. Alternatively, subunits and/or
subunit fragments are expressed in isolation. By expressing less than the full complement of subunits and
finding the combination that results in partially assembled complexes with properties expected of an intermediate structure, potential assembly intermediates can be identified. The yeast two-hybrid or protein overlay binding methods can screen for new proteins and
subunit regions that associate with a particular channel
subunit. All of these techniques are powerful means to
identify potential assembly intermediates, subunit regions, and new components in assembly. However, because they do not directly assay assembly, other methods must be used to validate the findings.
). Full assembly of an ion
channel has only been studied using cultured cells.
Even though cultured cells appear to be the system of
choice, they too are problematic. For example, with the
transient expression of heteroligomeric AChR subunits, <1% of the subunits assemble into AChRs because of cell-to-cell variations in the ratio of the subunit
cDNAs taken up by the cells (Eertmoed et al., 1998
).
This inefficiency in the assembly process prevents direct measurements of many aspects of the assembly
process. Nonetheless, cell cultures combine two critical
features: (a) high enough levels of expression to assay assembly, and (b) the expression of a set of chaperone
and processing enzymes required for assembly. Presently, the only kinetic assay used to study assembly in
cultured cells is pulse-chase metabolic labeling of subunits (Millar et al., 1996
; Trimmer, 1998a
). By first pulse-labeling subunits, and then following the labeled subunits, subunit folding and oligomerization can be assayed directly (e.g., Merlie and Lindstrom, 1983
; Schmidt
and Catterall, 1986
; Green and Claudio, 1993
).
). Certain nonionic detergents such as CHAPS and
digitonin tend be better than other detergents at preserving associations. Detergent-induced dissociation also
can be prevented by phospholipid-detergent mixtures
(Helenius and Simons, 1975
).
,
,
, and
that assemble into
AChR
2
pentamers. The consensus membrane topology of the AChR subunits is shown in Fig. 1. At the
amino terminal end, each subunit has a large extracellular domain that comprises approximately half the
subunit's mass. All ligand binding sites, N-linked glycosylation sites, and disulfide bonds lie within this domain. There are four transmembrane regions and a
large intracellular domain between the third and
fourth transmembrane regions. At the carboxy terminus, there is a short stretch of amino acids on the subunit's extracellular side just after the fourth transmembrane region. This membrane topology, as well as
other structural features, are shared by a family of neurotransmitter-gated ion channels that also includes neuronal AChRs, serotonin-3, glycine, and GABAA receptors, and it is likely that many aspects of AChR assembly are common to the whole family.
View larger version (14K):
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Fig. 1.
Consensus membrane topology of the AChR subunits.
M1-M4 represent putative transmembrane regions.
As shown schematically in Fig. 2, there are currently
two models that describe the assembly of ,
,
, and
subunits into the native AChR. In both models, assembly occurs along a pathway where a defined subset of
the four subunits assemble into intermediates that then
assemble into the
2
pentamer. In the first model (Fig. 2 A), the "heterodimer" model (Blount and Merlie, 1991
; Gu et al., 1991b
; Saedi et al., 1991
; Kreienkamp
et al., 1995
), the assembly is similar to that of HA in
that most subunit folding is completed before oligomerization can occur. Posttranslationally, the subunits
undergo a series of slow folding reactions before oligomerization, the best characterized being the formation
of the
-bungarotoxin (Bgt) binding site and the mAb
35 epitope on the
subunit. Afterwards, the "mature"
subunit associates with
or
subunits to assemble
or
heterodimers, and the heterodimers assemble
with
subunits into
2
pentamers. In this model, the two ACh binding sites, distinguishable by a difference in affinity for ligands such as d-tubocurare (dTC),
form on the
and
heterodimers. The evidence for
and
intermediates comes from studies where
and either
or
subunits were expressed in the absence of the other two subunits. Using steady state protocols, it was shown that heterodimeric complexes bind
Bgt and that binding is blocked appropriately by agonists and antagonists.
|
In the second model (Fig. 2 B), the "sequential"
model (Green and Claudio, 1993; Green and Wanamaker, 1997
, 1998
),
,
, and
subunits rapidly assemble
into trimers. The slow posttranslational folding of the
subunit occurs only after trimers are assembled. Soon
after the Bgt binding site forms, the
subunit joins the complex to make
tetramers. The first ACh binding appears on tetramers, after which the second
subunit is added to make
2
pentamers, and the second Bgt and ACh sites form on the pentamer. The evidence for this model is based on pulse-chase protocols
in which assembly intermediates were identified by
coimmunoprecipitation using subunit-specific antibodies, by immunoprecipitation with conformation-dependent antibodies, or by precipitation with affinity resin.
Once they are formed, most
trimers could be
"chased" into
tetramers, then into
2
pentamers, and finally onto the cell surface as
2
pentamers that demonstrated a precursor-product relation between each intermediate and the surface pentamers
(Green and Wanamaker, 1998
).
Although there are fundamental differences in the
two AChR assembly models, there are no disagreements about the data on which either model is based.
Similar data was obtained by all groups when cells expressing less than the full complement of subunits were
studied (see also Green and Claudio, 1993). Contradictions with the heterodimer model only arose when cells
expressing all four AChR subunits were studied. With
all four subunits present, two features of the methods
were critical in overcoming difficulties involved in isolating assembly intermediates. First, AChR complexes were solubilized in a detergent other than Triton-X 100 to prevent the dissociation of most AChR assembly intermediates (Green and Claudio, 1993
). Instead, subunit complexes were solubilized with a mixture of Lubrol PX and phosphatidylcholine. Second, the Torpedo AChR assembly is temperature sensitive (Claudio et al.,
1987
). When the temperature is lowered to 20°C, the
rate of assembly is slowed by more than an order of
magnitude, and the slow kinetics greatly aided in the
isolation of assembly intermediates. Although the Torpedo AChR subunits were used to obtain most of the
data in support of the sequential model, many features
of the sequential model were verified with the mouse
,
,
, and
subunits at 37°C (Green and Claudio, 1993
).
Ion Channel Subunit Associations and Folding
One difference between the two models is that in the
sequential model subunits rapidly associate into trimers
before most of the posttranslational folding occurs.
The associations are so fast that they could be cotranslational (Green and Claudio, 1993). K+ channel subunits
similarly associate rapidly, perhaps cotranslationally (Deal et al., 1994
; Shi et al., 1996
). If subunit associations occur cotranslationally, it is likely that the associating regions are at the NH2-terminal end of the subunits. This is consistent with studies that have shown
that regions near the NH2 terminus of AChR subunits
(Gu et al., 1991a
; Yu and Hall, 1991
; Sumikawa, 1992
)
and K+ channel subunits (Li et al., 1992
; Shen et al.,
1993
) mediate subunit associations. One reason why
subunits might associate so rapidly is to protect critical
domains from exposure to either the membrane or the
aqueous environment, which should help to prevent
misfolding of these domains.
Another difference between the two models is that,
in the sequential model, subunits continue to fold during subunit assembly and even after all of the subunits
have assembled together into pentamers. There is considerable evidence that subunit folding reactions occur
after each oligomerization step (Fig. 2 B). Furthermore, if a specific disulfide bond on subunits does
not form, assembly is completely blocked after assembly of
trimers. If the homologous
subunit disulfide bond does not form, assembly is blocked at a later
step, after assembly of
tetramers (Green and
Wanamaker, 1997
). Thus, the processing and folding
events between oligomerization steps are required for
assembly. The picture that has emerged from this work
is that subunit associations and folding during assembly
are continuous and interdependent processes. The assembly of the K+ channel appears to be similar in that
subunit folding occurs during and after the assembly of
the
subunit tetramer (Schulteis et al., 1998
). This
interdependence between subunit associations and
folding is shown schematically in Fig. 2 B by a major rearrangement of the trimer and tetramer subunits to allow for the insertion of unassembled
and
subunits.
Note that we know little about the structure of these assembly intermediates, and we are not proposing that
such a drastic structural change is actually occurring.
The available evidence indicates that AChRs (Smith
et al., 1987) and K+ channels (Nagaya and Papazian,
1997
) are assembled in the ER. However, it is uncertain
whether the late folding reactions occur in the ER since
it has been recently shown that subunit folding of AChR
and K channel subunits continues after formation of
the AChR pentamer and K+ channel tetramer. For both
AChRs (Green and Wanamaker, 1998
) and K+ channels (Schulteis et al., 1998
), the late folding appears to be required for normal channel function. Thus, late
folding events may serve to regulate whether channels
are in their functional state after release from the ER at
sites closer to where the channel will be targeted.
Conclusions and Perspective
Several features of ion channel assembly appear to be
different from that of other secretory pathway proteins.
Subunit associations can occur rapidly after synthesis,
and posttranslational folding and processing of subunits can occur throughout assembly, even after the final oligomeric complex is formed. That ion channel subunits continue to fold after associating with other
subunits should be considered when designing and interpreting experiments about channel structure and assembly. This is particularly relevant for experiments in
which less than the full complement of subunits are
studied or in which a subunit fragment is used to substitute for the full-length subunit. For example, the crystal
structure of the NH2-terminal K+ channel region, T1,
thought to mediate associations between subunits, was
obtained by studying the T1 fragment (Kreusch et al.,
1998). T1 formed a tetramer 20 Å in length with a central aqueous pore that was suggested to be the structure
of the channel's cytoplasmic vestibule. Another interpretation, however, is that this is a structure that forms
rapidly to initiate K+ channel assembly, and that the
subunits may continue to fold during assembly, ultimately forming a different structure.
Yet to be determined is the identity of the factors required for the assembly of ion channels in addition to
the subunits themselves. Presently, ion channels can assemble only in the environment of a cell. Some nicotinic receptors are properly assembled only in cells of
neuronal origin containing additional unidentified factors (Cooper and Millar, 1997, 1998
; Rangwala et al.,
1997
). Chaperone proteins are likely to be some of the
unidentified cellular factors required for assembly. The
ER chaperone proteins BiP (Blount and Merlie, 1991
;
Paulson et al., 1991
; Forsayeth et al., 1992
) and calnexin (Gelman et al., 1995
; Keller et al., 1996
, 1998
) associate with unassembled AChR subunits and may directly aid the assembly process. Interestingly, the proline isomerase, FKBP12, is a subunit in the functional
ryanodine receptor complex (Brillantes et al., 1994
).
Furthermore, NSF and
and
SNAPS associate with AMPA receptors in the dendrites of hippocampal pyramidal cells (Osten et al., 1998
) and appear to function
as "chaperones" that are required for channel function
or recycling (Nishimune et al., 1998
). These findings
raise important questions for future investigations about ion channel assembly. At what point does channel assembly end? Does it continue at the plasma membrane? Finally, can the folding and oligomerization
events that occur during assembly be distinguished
from conformational changes and protein associations that occur at the plasma membrane?
Original version received 16 November 1998 and accepted version received 21 December 1998.
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