(Received for publication, June 29, 1995; and in revised form, September 15, 1995)
From the
The potassium channel T1 domain plays an important role in the
regulated assembly of subunit proteins. We have examined the assembly
properties of the Shaker channel T1 domain to determine if the domain
can self-assemble, the number of subunits in a multimer, N and the mechanism of assembly. High pressure
liquid chromatography (HPLC) size exclusion chromotography (SEC)
separates T1 domain proteins into two peaks. By co-assembly assays,
these peaks are identified to be a high molecular weight assembled form
and a low molecular weight monomeric form. To determine the N
of the assembled protein peak on HPLC SEC, we
first cross-linked the T1 domain proteins and then separated them on
HPLC. Four evenly spaced bands co-migrate with the assembled protein
peak; thus, the T1 domain assembles to form a tetramer. The absence of
separate dimeric and trimeric peaks of assembled T1 domain protein
suggests that the tetramer is the stable assembled state, most probably
a closed ring structure.
Voltage-gated K channel proteins are
multisubunit ion channel proteins. The core channel consists of an
apparent tetramer of
-subunit proteins that assembles to form the
K
ion-selective aqueous pore across the cell's
plasma membrane(1) . In addition,
-subunit proteins, which
are apparently not transmembrane proteins, can be attached to each
-subunit protein(2) . Molecular cloning has revealed a
large diversity of K
channel subunit proteins.
Sequence comparisons among
-subunits has revealed that the
similarities among encoded proteins cluster into a variety of
K
channel subfamilies(3) . Biophysical studies
have indicated that these subfamilies are in fact functional subsets of
channel proteins in that functional heteromultimeric channels have only
been formed by co-expression of two
-subunit proteins from the
same subfamily(4) .
The mechanisms that govern the assembly
and function of voltage-gated ion channels are poorly understood.
Recently we and others have identified a conserved molecular domain,
the T1 domain, encoded within the cytoplasmic N terminus of the
-subunit protein that plays an important role in the assembly of
K
channel subunit
proteins(5, 6, 7, 8, 9, 10) .
Our studies have suggested that the T1 domain, translated by itself,
can self-assemble(6) . Sucrose density gradients reveal the
formation of a high molecular weight complex; co-immunoprecipitation
studies show that a tagged T1 domain protein can co-precipitate another
un-tagged T1 domain protein. In addition, the T1 domain contains the
molecular recognition sequences required for the subfamily-specific
assembly of voltage-gated K
channel
proteins(11) . Chimeras made with swapped N-terminal sequences
show the assembly specificity of the N-terminal donor; the soluble T1
domain translated by itself only co-assembles with T1 domain proteins
made from the same subfamily. These results have prompted our
hypothesis that the T1 domain is the primary site for organized
tetramerization of K
channel subunit proteins along
subfamily-specific lines.
Other recent studies have questioned the
role that the T1 domain plays in K channel subunit
assembly. Subunit proteins synthesized with deleted N termini have been
shown to function in Xenopus oocyte expression
systems(9, 10, 12, 13) . In
addition, another group has suggested that S1, the first transmembrane
domain, is necessary for assembly, based on their hydrodynamic and
co-precipitation studies(7) . In fact, no studies have directly
examined the assembled T1 domain protein to show that it is forming a
tetramer on its own. Tetramerization has only been inferred based on
the migration rate of the protein in sucrose gradients or gel
filtration columns. In addition, the molecular species responsible for
the co-immunoprecipitation signal has not been identified. It is not
clear to what extent dimers, trimers, tetramers, larger octomers, or
complexes with other proteins contribute to the co-immunoprecipitation
signal. Finally, what role the T1 domain plays in the normal assembly
of other, non-Shaker type potassium channels has not been directly
examined.
In this paper, we have developed methods to efficiently
separate T1 domain proteins into different molecular weight species, in
order to separate assembled and unassembled proteins. By combining
molecular size separation with co-precipitation techniques we have
determined the assembled species that are responsible for the
co-precipitation of untagged T1 domains. Finally, using chemical
cross-linking we have measured the number of subunit proteins assembled
in the peak assembled fraction. These results have allowed us to
formulate more specific models of the mechanisms by which the T1 domain
functions in K channel assembly.
In vitro translation samples were prepared by mixing 1-2 µl of in vitro translation reaction with 200 µl of bind buffer.
The sample was then cleared by centrifugation at 60,000 rpm for 20 min
at 4 °C in a mini-ultracentrifuge (Beckmann). For HPLC samples,
individual fractions were adjusted to bind buffer conditions, by the
addition of salts, CHAPS, and imidazole, before applying to the resin.
Sample supernatant was mixed with the resin for 10 min at 4 °C to
ensure complete binding of poly-His-tagged proteins to the resin.
Unbound material was then spun through the column, and the resin was
washed in a series of steps: first 1 wash with 350 µl of
bind buffer, followed by 3
washes with 350 µl of wash
buffer (bind buffer with 20 mM imidazole). Samples were eluted
in 350 µl of elution buffer (bind buffer with 1 M imidazole). All fractions were collected and analyzed by SDS-PAGE
gel and autoradiography. Samples were concentrated for gel analysis
either by acetone precipitation or immunoprecipitation with the common
anti-1B antiserum(11) .
For HPLC analysis, samples were cross-linked at 1.5 mM DSS, as described above, and then injected into the HPLC following quenching.
HPLC runs were
standardized by detection of protein standards using an absorbance
detector set at 280 nm, before a series of experimental runs were
performed. Because of the small amount of synthesized protein relative
to the total protein in the in vitro translation mix, T1
domain proteins were not directly detected by UV absorbance but rather
by S autoradiography of SDS-PAGE gels run on the
fractionated column eluate. To calculate the distribution coefficient, K
, which describes the average access of a protein
to the total column volume, we needed to measure the void and total
volumes for the columns. Void volume was detected as the first
appearance of protein aggregates; the total volume was detected by the
appearance of vitamin B-12. K
is (peak volume
- void volume)/(total volume - void volume)(14) .
For in vitro translated proteins, peak protein fraction
volumes were set at the midpoint volume for the fraction and normalized
back to the absorbance detector by subtracting the volume in the line
after the detector up to the fraction collector. In internal control
for this correction was the peak hemoglobin fraction. Protein peaks
that were evenly divided between two fractions were considered to peak
at the point where the fraction changed. Because these K
measures for T1 domain proteins were only accurate to half a
fraction volume, these numbers are considered approximations.
Soluble N-terminal peptides from the Shaker type K channel subunit protein AKv1.1, containing the T1 domain (see Fig. 1) were synthesized in vitro using a rabbit
reticulocyte lysate system, as described
previously(6, 11) . All proteins translated
efficiently without the addition of microsomal membranes and were
soluble without added detergent. Full N-terminal peptides that were
studied include CF2-Tag1, an epitope tagged N-terminal
peptide(6) ; 1ABC(no tag), the full N-terminal protein with
restriction sites added between subdomains of the T1
domain(11) ; and 1ABC(poly-His), the 1ABC protein cloned in
frame with the C-terminal 6-His sequence of the pCITE-2A vector
(Novagen). In addition, two constructs encoding the Shaker T1 domain
with minimal additional sequences were examined: the epitope-tagged T1
domain protein, 1T1-T7tag(11) , which uses the pCITE-2A
initiator Met, and 31A1BC(11) , which uses the AKv3.1a
initiator Met and 10 amino acids, before the Shaker T1 domain. There
were no detectable differences in the behavior of any of these
constructs except for epitope tag sensitivity and longer retention of
T1 only proteins on SEC HPLC due to their smaller size. The results
presented are based on over 110 individual HPLC runs, with a minimum of
three experiments for any individual result.
Figure 1:
Schematic
description of the Shaker T1 domain containing constructs that were
used in these experiments, showing the region of the parent AKv1.1a
clone that these constructs originated from. Full N-terminal constructs
are CF2-Tag1 (AKv1.1a, amino acids 1-193), 1ABC, and
1ABC(poly-His) (AKv1.1a, amino acids 1-196). T1 domain only
constructs are 1T1-T7tag (AKv1.1a, amino acids 58-196) and 31A1BC
(AKv3.1a, amino acids 1-10, followed by AKv1.1a, amino acids
67-196). Locations of the conserved subdomains A, B, and C of the
T1 domain are indicated, as well as the locations of the epitope tags
used and the restriction enzyme sites that are introduced into the
clones to facilitate chimera construction (see (11) ). The
first transmembrane domain, S1, begins at AKv1.1a,
Leu.
Figure 2:
T1 domain proteins separate into two peaks
on size exclusion chromatography columns. In vitro translated
1ABC protein was separated on a Bio-Silect 250-5 (Bio-Rad)
silica-based size exclusion HPLC column. This columns separates
proteins based on the size of the molecule, with larger proteins
eluting in earlier fractions. A, autoradiography of HPLC
fractions showing the separation of
[S]Met-labeled 1ABC protein through this column.
The first fraction run on the gel is the void volume; the last fraction
is the total volume. B, quantitation of the autoradiograph shown in panel A. Band volumes were measured for 1ABC
protein in each fraction and plotted versus the fraction
number. As is clear from the autoradiograph and the quantitation, the
1ABC protein separates into two distinct populations of differing
apparent size during the chromatography.
Figure 3: Co-immunoprecipitation analysis of HPLC protein peaks identifies assembled fractions. CF2-Tag1 and 1T1-T7tag were co-translated then run on a Bio-Silect 400-5 SEC column to separate according to size. Fractions corresponding to the positions of the high molecular weight peak (fractions 19, 20, and 21) and the low molecular weight peak (fractions 23, 24, and 25) were pooled separately. Both peaks and the original unfractionated translation were probed to determine if the proteins were co-assembled. Anti-1B is a common antiserum to the B subdomain of the T1 domain in both proteins, and it is a positive control for the presence of both proteins(11) . Anti-Tag1 only recognizes CF2-Tag1; thus co-precipitation of 1T1-T7tag by this antiserum is indicative of assembly. The minus antibody lane(-) is the negative control. Both proteins are found in the two peaks separated on SEC HPLC, as verified by precipitation with anti-1B; however, 1T1-T7tag is not co-precipitated in the low molecular weight peak. Thus only in the high molecular weight peak are the two proteins co-assembled.
To determine which peak(s) contain assembled proteins, we co-translated 1T1-T7tag, and CF2-Tag1 and separated the proteins on a Bio-Silect 400-5 SEC HPLC column. High molecular weight and low molecular weight fractions were separately pooled, and assembly-tested in the two protein populations by anti-Tag1 antiserum immunoprecipitation. Both 1T1-T7tag and CF2-Tag1 proteins are present in both peaks, as verified by immunoprecipitation with the common anti-1B antiserum. However, 1T1-T7tag proteins are only co-immunoprecipitated from the high molecular weight protein fractions and not from fractions corresponding to the low molecular weight peak. Therefore, the proteins in the low molecular weight peak are not co-assembled and thus are probably monomeric. Identical results were obtained from proteins separated on Bio-Silect 250-5 and 125-5 SEC columns.
Figure 4:
Ni affinity column
co-purification analysis demonstrates T1 domain protein assembly. T1
domain proteins were in vitro translated and then applied to
Ni
affinity columns. The columns were washed at
increasing stringency, and then bound proteins were eluted at a high
imidazole concentration (1 M imidazole). A, affinity
chromatography of 1ABC(no tag) on Ni
affinity resin. L, sample loaded; F, column flow-through, B,
bind buffer wash. W, washes with wash buffer. E,
elution with elution buffer. 1ABC(no tag) does not bind to the
Ni
affinity resin; therefore, no protein is present
in the elution step. B, affinity chromatography of 1ABC(no
tag) co-translated with 1ABC(poly-His) on Ni
affinity
resin. Although 1ABC(no tag) does not directly bind to the
Ni
affinity resin, it is present in the elution step
because of its stable assembly with 1ABC(poly-His), which does bind to
the column.
NiColumn Analysis of Proteins Separated on
HPLC-The Ni
affinity column
co-purification analysis was next applied to fractions from the HPLC
separation of co-translated 1ABC(no tag) and 1ABC(poly-His). The raw
fractions, before application to the Ni
affinity
resin, show that both proteins are separated in similar manners,
suggesting that the proportion of assembled and unassembled proteins
was similar for each. The fractions were applied to Ni
affinity columns, washed, and eluted. Elution off the
Ni
column shows that 1ABC(poly-His) proteins are
purified identically to their original profile; however, 1ABC(no tag)
proteins are only co-purified in the higher molecular weight fractions.
Quantitation of the counts for the various proteins, following
Ni
affinity column co-purification analysis on the
different fractions, shows that 1ABC(no tag) T1 domain protein
co-purifies as a monophasic peak that is identical to the high
molecular weight peak identified above (see Fig. 5). This
suggests that the high molecular weight peak is the sole form of stably
assembled T1 domain proteins and the low molecular weight peak is
completely composed of monomeric proteins.
Figure 5:
Ni affinity column
co-purification analysis of HPLC fractions shows assembled protein
separation profile. 1ABC(no tag) and 1ABC(poly-His) were co-translated
and separated on a Bio-Silect 250-5 SEC HPLC column. Fractions
from the column were split in half; half the fraction was analyzed for
the protein migration, and the other half was applied to a
Ni
affinity column. A, autoradiography of
proteins separated on a Bio-Silect 250-5 SEC column. B,
band volume quantitation of experiment in panel A. The
co-translation autoradiograph shows the chromatography of the T1 domain
proteins before application to the Ni
affinity
columns. OT lane shows the unfractionated translation. The
following lanes are successive fractions from the column,
starting at the void volume. Upper band is 1ABC(poly-His), and
the lower band is 1ABC(no tag). Both proteins are present in
the translation and show essentially identical SEC separation patterns
with high and low molecular weight protein peaks. The Ni
column flow-through for the HPLC fractions shows only a single
protein at the 1ABC(no tag) molecular weight. In the Ni
column elution, both 1ABC(no tag) and 1ABC(poly-His) are present
in fractions corresponding to the high molecular weight peak; however,
only 1ABC(poly-His) is present in the fractions corresponding to the
low molecular weight peak. Therefore, T1 domain proteins in the
assembled state are completely contained within the high molecular
weight peak fractions.
, 1ABC(poly-His):Ni
column eluted;
, 1ABC(no tag):Ni
column
eluted;
, 1ABC(no tag):Ni
column
flow-through.
Figure 6: Chemical cross-linking of T1 domain proteins. 1ABC was in vitro translated and then cross-linked with increasing concentrations of DSS, as described under ``Experimental Procedures.'' Cross-linked proteins were run on a 5-20% linear gradient SDS-PAGE gel, and the positions of the proteins were determined by autoradiography. The first lane shows the migration of molecular mass standards. The monomer band is the only band present in the non-cross-linked sample. At the lowest level of cross-linking, a band at the expected position for a dimer is seen. At higher cross-linker concentrations, bands are seen at the expected positions for the trimer and tetramer, with additional material present as a diffuse smear at high molecular weights. The protein band at the tetramer position is broader than the other bands and appears to be a doublet.
Figure 7: SEC HPLC analysis of cross-linked T1 domain proteins characterizes the assembled protein peak. 1ABC was in vitro translated, then cross-linked with 1.5 mM DSS as described under ``Experimental Procedures.'' This cross-linked material was then separated by size on a Bio-Silect 250-5 SEC HPLC column. Fractions were acetone-precipitated and then run on a 5-20% linear gradient SDS-PAGE gel and subjected to autoradiography to determine the migration pattern of the different protein species. A, autoradiograph of the HPLC fractions following separation of cross-linked 1ABC. B, densitometry analysis of bands from the gel in panel A. Band volume for proteins at the different molecular weight sizes was measured and plotted without normalization versus the fraction number. The monomer band shows two peaks at the expected high and low molecular weight positions seen in uncross-linked samples. Dimer, trimer, and tetramer bands all show a single peak in the fraction corresponding to the high molecular weight peak fraction for uncross-linked samples. The higher molecular weight smear of cross-linked protein peaks in earlier fractions that do not correspond to the position of protein peaks in uncross-linked samples. Therefore, the high molecular weight T1 domain peak is identified as being composed of T1 domain protein homotetramers.
The SEC HPLC separation of the proteins shows that T1 domain proteins can be cross-linked up to the tetramer size and still migrate in the high molecular weight peak, thus identifying the higher molecular weight peak as the tetrameric form of the protein. Furthermore, the fact that the dimer and trimer proteins also show a single peak at the tetramer position indicates that they are partially cross-linked tetramer proteins that are separated during the SDS-PAGE analysis.
The T1 domain was originally identified through its role in
K channel subunit assembly(5, 6) .
Later, it was shown to be a critical regulator of subunit
heteromultimerization, by controlling the subfamily specific
heteromultimerization of K
channel subunit
proteins(11) . This work has sought to examine the T1 domain,
separate from the K
channel transmembrane domains, to
measure directly the self-assembly properties of the T1 domain.
HPLC analysis shows that the T1 domain exists predominantly in two forms, a high molecular weight form and a low molecular weight form. Both co-immunoprecipitation and cross-linking analysis clearly identify the high molecular weight form as an assembled state of the protein and the low molecular weight form as the unassembled, monomeric form of the T1 domain. Chemical cross-linking shows that all the assembled T1 proteins cross-link in an evenly spaced ladder; therefore, the T1 domain can assemble as a homomultimer. Four different sizes of T1 domain protein are produced by cross-linking, monomer, dimer, trimer, and tetramer. Larger molecular weight cross-linked complexes run in anomalous positions on HPLC and therefore are probably artifactually created during the cross-linking procedure. Dimer, trimer, and tetramer bands all show a single peak on HPLC that lines up with the assembled peak seen on uncross-linked samples. Therefore, these structures represent variable cross-linking of a single, tetrameric assembled form of the protein.
The lack of a detectable binary assembled form in the T1 domain HPLC profile suggests that there is no particular stability associated with this structure. This observation argues against subunits assembling as dimers and the dimers assembling as tetramers in an isologous association. Rather, the results suggest that T1 domain proteins assemble by the monomeric addition of single subunit proteins in a repeated A-B interface interaction pattern until the fourth subunit protein completes the closed ring. Such heterologous association has been observed in the multimerization of other membrane proteins, such as neuraminidase(15) .
Closer analysis of the
cross-linked tetrameric band, following autoradiography of SDS-PAGE
gels reveals that it often appears to be a doublet or a broader band
than any of the other T1 domain bands. This suggests that the tetramer
can be cross-linked into two different structures. One structure is
presumably the linear form, common to the monomer, dimer, and trimer
cross-linked proteins. The second structure probably represents the
fully cross-linked, native form of the assembled T1 domain. Based on
the presumed form of the functional K channel, with an
axis of symmetry down a central aqueous pore, and the unique stability
of the fully assembled state of the T1 domain, the native form of the
assembled T1 domain is probably a closed circular, ring, structure.
A closed circular structure of the assembled T1 domain would provide
additional stability to the tetramer by providing stabilizing
subunit-subunit interactions on both sides of the T1 domain, thus
requiring two separate interactions to be broken in order to disrupt
the structure. Indeed, co-purification analysis on HPLC fractions,
using a poly-His-tagged subunit protein co-translated with an untagged
subunit protein, shows that co-purification is limited to the tetramer
peak. Thus interactions between T1 domain proteins that are not
completed to the tetrameric state must dissociate within the time frame
for these experiments. Cross-subfamily interactions between T1 domains
or point mutations that disrupt the tetramer may be filtered out by
this stringent assembly requirement. Selectivity against assembly of
K channel subunit proteins across subfamilies or
disruption of assembly by mutagenesis can therefore occur either
through incompatible interface amino acids or through subtle structural
differences between proteins with compatible interfaces that prevent
the closing of the ring, either through torsion or interface angle
incompatibility.