(Received for publication, March 20, 1995; and in revised form, June 12, 1995)
From the
We have raised antisera against recombinant peptides expressed from cDNAs fragments common to all splicing variants generated at the Shaker locus of Drosophila and used them as a tool to biochemically characterize these channel proteins. This antisera succeeded in detecting the expression of multiple Shaker potassium channels (Sh Kch), proteins with variable molecular mass (65-85 kDa) and pI (5.5-7). Additionally, for first time, specific Sh proteins of 40-45 kDa most probably corresponding to some of the so-called short Sh cDNAs previously isolated by others have been identified. Using genetic criteria, it has been determined that at least a good part of this variety of proteins is generated by alternative splicing. Developmental experiments show a double wave of Sh Kch channel expression with a first pick at the third instar larvae stage, a minimum at the beginning of puparation, and the highest plateau 36 h after hatching of adult flies. The pattern of Sh splice variants changes dramatically throughout development. A detergent-resistant fraction with about 50% of Sh Kch which seems to be anchored to submembranous structures has been found. Finally, other biochemical properties of Sh Kch, like membrane fractionation and glycosylation, are also described.
Potassium channels are the molecules mostly responsible for stabilizing the membrane potential of excitable cells thereby regulating their excitability(1) . Most cells express various potassium channels with diverse functional and pharmacological properties(2) . Thus, in the nervous system different types of potassium channels set the resting potential, control the duration and frequency of action potentials, determine the length of bursting periods, etc. In contrast to other ionic channels, this diversity and ubiquity, together with their small quantity, and the lack of good molecular markers have precluded the productive standard purification of most voltage-gated potassium channel proteins. Thus, the first cloning of a potassium channel gene was undertaken by means of a genetic approach(3, 4, 5) . Homology cloning in different species and tissues together with site-directed mutagenesis and in vitro expression have unravelled the existence of various voltage-gated potassium channel gene subfamilies and yielded a great increase in the knowledge of these molecules (see (6) and (7) for recent reviews). In spite of this great advance in molecular studies and a few brave purifications (8, 9, 10, 11) , the above mentioned difficulties have prevented successful investigation into the biochemical properties, tissular and cellular distribution, and other relevant data regarding the potassium channels proteins.
The Shaker locus of Drosophila harbors a transcription
unit that by means of alternative and differential splicing at its 3`
and 5` ends can produce a wide variety of transcripts encoding
homologous proteins. The resulting channel isoforms share a common
central core region and have different N-terminals and C-terminals (12, 13, 14) which confer differential
electrical properties when expressed in Xenopus oocytes(15, 16) . It has been shown that four Sh ()subunits assemble to form a functional potassium
channel(17) . Since properties of heteromultimeric Sh Kch
differ from those of homomultimeric channels(18) , the possible
combination of Sh isoforms can provide in this way a molecular basis
for potassium channel diversity in this organism.
Although, there have been previous reports on the preparation of antisera against Shaker proteins from Drosophila(19, 20) , the theoretically expected variety of Sh Kch isoforms was not detected and the biochemical study of these proteins was not followed up.
We show here that by using immunochemical techniques the biochemical characterization of native Shaker potassium channels from Drosophila can be successfully approached.
Western blot was done by electrotransferring the proteins of the gel to nitrocellulose membranes under standard conditions. Immunoblots were carried out with monospecific anti-Sh antisera and a secondary goat anti-rabbit IgG antiserum couple to horseradish peroxidase. Finally, the chemiluminescent ECL method (Amersham) for the detection of antibody binding was used. When required, laser densitometry and the computer program Image-Quant (Molecular Dynamics) were used for the quantitation of immunoblots. The optical density obtained in this way was standardized against the total protein applied to the gel which was calculated by Coomassie Blue staining and densitometry of a gel run in parallel. All quantitative experiments were done at least in duplicate and repeated with two different sets of samples. Results are shown as average.
We have raised affinity purified polyclonal antisera against
various recombinant proteins obtained by expression of different cDNA
fragments of the central core region common to all Sh transcripts. All
of them recognized several specific Shaker proteins but after
a selection procedure (see ``Experimental Procedures''), one
of them, called -ShHX1, showed the highest titer and affinity and,
therefore, was used in all the experiments described in this paper.
An analysis by Western blot of total proteins from adult flies with
-ShHX1 detects multiple bands with apparent molecular mass in the
range of 65-85 kDa (Fig. 1A). The molecular size
of the detected peptides is slightly larger than the theoretical
molecular mass (65.1-76.2 kDa). All the labeled proteins must be
specific to Shaker since the antiserum is monospecific and,
moreover, these bands are absent in the track of control Df flies.
Almost all the protein detected with the antiserum is in the P3
membrane fraction. None of the bands seemed to appear in significant
amounts in the cytosolic S3 extract (Fig. 1B), probably
indicating the low occurrence or total absence of any intracellular
pool of channels as that described for sodium and calcium
channels(30, 31) . The results presented here are in
apparent contradiction to the large intracellular pool found in the
case of Shaker-baculovirus infected insect cells (32) although, as the authors pointed out, this might be a
consequence of hyperexpression. High resolution bi-dimensional
electrophoresis and Western blot dramatically improves the resolution
of immunolabeled bands. Thus, a homogenate of head proteins yields more
than 10 spots with different pI and molecular mass in the range of
65-85 kDa (Fig. 1C). Again, all the immunostained
spots seem to be specific since none of them is detected in the Df
flies blot (Fig. 1D). Very interestingly, a single spot
of about 45 kDa at the middle of the pI range is also specifically
labeled (Fig. 1C, arrowhead). Due to various
reasons it is very unlikely that the observed multiplicity of bands
could be generated by degradation of channel proteins. First, the
denaturing conditions used in the preparation of samples (see
``Experimental Procedures'') strongly preclude this
possibility. Second, when a homogenate of head proteins prepared for
bi-dimensional electrophoresis was left at room temperature for up to 5
h prior to being applied to the gel, no significant changes in the
relative intensity of spots were observed (not shown), clearly
indicating that the various proteins detected are not the result of
proteolysis during sample preparation. Due to its size, the 45-kDa
protein might correspond to one of the so-called short Shaker cDNAs isolated in a few
laboratories(3, 12, 13) . We have checked
whether this short Shaker protein is in fact inserted in the
plasma membrane. To this end, a protein extract of a membrane
preparation from adult head (Fig. 1E) was analyzed.
Except for some changes in the most acidic proteins, probably due to
modifications in the phosphorylation state of the channel proteins
commonly produced during the isolation of the membranes, the pattern of
membrane proteins is very similar to that of total proteins. This
experiment shows that the 45-kDa peptide is indeed in the plasma
membrane.
Figure 1:
Western blot analysis of Sh
potassium channels proteins. Total proteins from adult whole flies (A), heads (C, D, F, G, and H), and
S3-cytosol (B) or P3-membrane (B and E)
fractions of wt or mutant as indicated, were electrophoresed
in either mono-dimensional or bi-dimensional gels, electroblotted, and
probed with -ShHX1 antiserum. Complete blots are shown for
mono-dimensional electrophoresis while in the case of bi-dimensional
blots, panels show only the area corresponding to approximately
molecular mass = 35-95 kDa and pI 5.5-7 where all
the specific spots were detected. Exposure time in the autoradiography
ranged from 2 to 5 min. G, total head proteins were analyzed
by a modification of the electrophoretic technique of Kite and
Rodriguez (26) as indicated under ``Experimental
Procedures.'' H, to the same sample as that load in C, we added SDS to a final concentration of 2% and the sample
was incubated at 65 °C for 15 min. Finally, proteins were analyzed
by regular mono-dimensional SDS-PAGE and Western
blot.
In order to check whether this variety of protein detected
by our antiserum is in fact generated by alternative splicing at the Shaker locus, a genetic approach has been taken. Previously,
by using various recombinant Sh proteins containing the central common
core region and different alternative amino and carboxyl ends, it has
been found that antiserum -ShHX1 is able to equally recognize all
Sh Kch isoforms (not shown). The chromosomal break point W32 interrupts
the Shaker transcription unit between exon 2 and 3 (13) but does not completely preclude the
transcription(14) , probably through the use of a second
promoter located downstream of the translocation point. Therefore, at
least exons 1 and 2 cannot be transcribed and the corresponding
alternative splice transcripts ShB and ShD (in the nomenclature of (13) ) are never produced in this mutant. As expected, the
bi-dimensional immunoblot pattern of W32 flies lacks a few of the
peptides labeled in wt flies (Fig. 1, C and F) clearly indicating that the complex pattern of Sh proteins
is indeed generated by alternative splicing. The fact that short Sh
proteins also disappear in W32 flies, gives clear evidence for its
specificity and additionally indicates that the 45-kDa peptide must
belong to either ShB or ShD N-terminal isoforms. A couple of very close
spots running at equal molecular size on the most acidic side of the
gel are not detected in this mutant either (Fig. 1C,
arrows), whereas another protein on the most basic side of the W32
map increases its intensity in respect to CS (Fig. 1F, arrowhead). Thus, the question
remains as to why this smaller 45-kDa Sh peptide could not be detected
by mono-dimensional SDS-PAGE and Western blot (as in Fig. 1A, for instance). One explanation could be the
differences in the manner of preparing samples for each type of
electrophoresis. Accordingly, a modified mono-dimensional
electrophoretic method which uses concentrated urea instead of SDS as
denaturating agent (26) was used. Under these experimental
conditions an intense band of 45 kDa and a weaker one of 41 kDa
together with a few intense bands in the range of 65-90 kDa were
specifically labeled (Fig. 1G). Moreover, when an
urea/Nonidet P-40 protein extract as that used for bi-dimensional
electrophoresis of CS head proteins (Fig. 1C)
was heated in the presence of SDS and run in a regular SDS-PAGE gel,
peptides in the range of 65-85 kDa were normally labeled while
the 45-kDa protein was no longer detected (Fig. 1H).
Altogether, these results point to a probable aggregation and
precipitation of the short Sh peptides during boiling with SDS. This
can also explain why these truncated channel proteins have not been
detected in previous work by other
laboratories(19, 20) .
A quantitative immunoblot analysis of the anatomical distribution of Sh Kch has been carried out. As shown in Fig. 2A, Sh proteins are more abundant in the head than in thorax, and they are practically absent in the abdomen of adult flies. This distribution of Sh Kch proteins correlates well with that of nervous tissue. Additionally, with this simple anatomical dissection one can observe that the pattern of Sh proteins of head and thorax are rather different (Fig. 2C), clearly indicating a tissue-specific alternative splicing as previously suggested by Northern blot analysis(14) .
Figure 2:
Quantitative immunoblotting of different
tissue homogenates. A, quantitative analysis of the
immunoblots (see ``Experimental Procedures'') of the CS tracks in panel B carried out with affinity purified
-HXSh1 antiserum. B, detail of the central part of an
immunoblot of total proteins from head, thorax, and abdomen of CS and Df flies electrophoresed as in Fig. 1A. C, further detail at higher magnification of head and thorax
tracks of CS. Notice that in order to compare both band
patterns the thorax autoradiography was overexposed to bring it up to
about the same intensity as that of head.
Protein extracts
of developmentally staged samples of Drosophila have been
analyzed by quantitative immunoblotting with the antiserum -ShHX1.
As shown in Fig. 3A, there are two waves of Sh Kch
expression during development. Sh proteins are first clearly detected
in first instar larvae and increase in intensity up to the early third
instar. At the beginning of puparation they seem to disappear. The
expression restarts at the late pupal stage and continues to increase
up to the adult fly. Changes in the pattern of bands can be nicely
viewed in bi-dimensional blots. Thus, protein extracts from larval
brain (Fig. 3B) were prepared and their bi-dimensional
immunoblot was compared to that of adult head (Fig. 1C)
since both structures hold more than 90% of the Sh proteins present in
their respective developmental stages. The pattern of adult brain is
clearly more complex than that of larval central nervous system. This
lacks most of the high molecular size proteins and shows no more than
four labeled spots that seem to have their counterpart in the adult
bi-dimensional map including a weak labeling of the 45-kDa spot. As
stated above, immunoblots carried out from mono-dimensional gels under
normal conditions did not detect a significant amount of Sh channels in
protein extracts from embryo (Fig. 3A). In situ hybridization with a variety of probes has also failed in
detecting Sh transcripts in embryos(33) . However, Sh potassium
channel currents have been recorded in embryonic cells(34) .
Therefore, we have tried to improve the detection level by overloading
bi-dimensional gels and increasing the time of autoradiography in the
final chemiluminescent reaction of the immunoblot. Under these
experimental conditions Sh proteins could finally be detected (Fig. 3C) indicating that even at considerably lower
comparable levels Sh Kch are indeed expressed in embryonic stages. The
bi-dimensional map obtained in this way is very similar although not
identical to that of larval central nervous system and mainly consists
of the most acidic high molecular mass proteins and the 45-kDa spot
(compare Fig. 1C and Fig. 3, B and C). It must be highlighted here that the Sh proteins most
abundant in embryo and larval brain are those which disappear in W32
flies (ShB and ShD as above). A density separation of membranes from Drosophila head has been carried out by using
ultracentrifugation in stepped Ficoll gradients and the distribution of
Sh Kch was analyzed as shown in the diagram of Fig. 4A.
Although Sh Kch are more concentrated in the 8-12% fraction, all
membrane fractions isolated from the various interfaces contained
considerable amounts of Sh Kch proteins. This separation of membranes
does not seem to yield any segregation of Sh Kch isoforms since these
different membrane fractions exhibit a rather similar mono-dimensional
immunoblot pattern (Fig. 4A). Although very much alike
techniques have been applied to the isolation of Drosophila neuronal membranes (29) , the lack of prevalence of
different Sh proteins that was observed in the fractionation of
neuronal membranes could be due to an inefficient separation. In order
to check this point, the morphological composition of membrane
fractions has been analyzed with EM techniques. The EM micrograph of
total P3 membranes shows a very heterogeneous composition of vesicles
of different shape, size, and appearance (Fig. 4B). On
the contrary, membrane fractions purified by density
ultracentrifugation exhibited a more homogeneous membrane composition
than the crude membranes but each was different from the others. For
instance, the 8-12% interface fraction, containing the highest
concentration of Sh proteins, is quite rich in vesicles of 80-100
nm in diameter as well as highly electrodense walls (Fig. 4C, arrows). Altogether, these experiments
demonstrate that the major Shaker proteins are more or less
evenly distributed in fractions of nervous tissue membranes of diverse
compositions. However, with this kind of analysis the possibility that
Sh isoforms with a low level of expression might be segregated in
different kinds of membranes cannot be ruled out.
Figure 3: Analytical and quantitative immunoblot of developmentally staged samples. A, quantitative determination of Sh proteins in developmentally staged samples carried out as described elsewhere. The data was standardized to the value of A48. Abbreviations are: E, embryo; LI, first instar larvae; LII, second instar larvae; ELII and LLII, early and late third instar larvae; EP and LP, early and late pupae; A0-48, adult flies collected at the indicated time (hours) after emerging. B and C, bi-dimensional immunoblot of larval brain and embryo protein extracts, respectively. Electrophoretic conditions are the same as in Fig. 1C. Autoradiography exposure times were 20 and 45 min, respectively.
Figure 4: Distribution of Shaker potassium channels in purified membranes. A, total membranes of Drosophila head (P3) were fractionated in discontinuous Ficoll gradients as described under ``Experimental Procedures.'' Membranes were collected from density interfaces as indicated in the scheme and either used for electrophoresis and immunoblotting (lower panel) followed by quantitative determination of Sh proteins (upper panel) or for EM analysis (C and D). The concentration of Sh Kch to total membrane protein has been standardized to the value of the 8-12% fraction because this contains the highest concentration. The low amount isolated from the 16-20% interface preclude any accurate analysis of this fraction. B and C, EM micrographs of the membranes pelleted from P3 and the 8-12% fraction, respectively. Pictures were taken at magnification 25,000. Arrows point to a population of membrane vesicles enriched in that fraction. Bars correspond to 200 nm.
In order to further biochemically study the Sh Kch proteins, membrane fractions were solubilized. Most common biological detergents tested, including CHAPS, deoxycholate, Nonidet P-40, and Triton X-100, were efficient enough in solubilizing these channel proteins. As can be seen in Fig. 5A, increasing concentrations of detergents progressively solubilized Sh protein, reaching a plateau at around 50% of the channel proteins. Addition of either the same or other detergents to the insoluble fraction did not solubilize further significant amounts (Fig. 5B). This has lead to the conclusion that the detergent-resistant fraction of Sh Kch is the same for all nondenaturating detergents. In order for the solubilization to be completed, strong denaturating agents such as urea (Fig. 5B) or boiling in SDS had to be used. It must be also mentioned here that both solubilized and insoluble fractions have a very similar pattern of bands regardless of the detergent and concentration used (Fig. 5C), probably indicating that at least the most abundant Sh Kch isoforms are present with similar ratios in both fractions.
Figure 5: Solubilization of Shaker potassium channels proteins. A, total membranes of Drosophila head were solubilized with increasing concentrations of either deoxycholate or Triton X-100. Solubilized and insoluble fractions were analyzed by electrophoresis and Western blot and the relative amounts of Sh proteins determined by densitometry as explained elsewhere. B, sequential solubilization of membranes with two detergents were also carried out as schematically shown. Concentrations of detergents and other compounds are as indicated. The rest of experimental conditions are as those used for solubilization in a single step. C, detail at high magnification of a mono-dimensional immunoblot containing soluble and insoluble fractions generated with 0.3% deoxycholate.
Other well known voltage-sensitive
channels such as sodium channels have shown a wide variety of
post-translational modifications(35) . Here the biochemical
characterization of post-translational modifications of Sh Kch has been
initiated by studying its glycosylation. Because of probable
interference of deoxycholate with some glycosydases, Triton X-100
soluble fractions were used for this purpose. As shown in Fig. 6, N-glycopeptidase F produces some sharpening and
a clear shift of about 6 kDa to lower M in all
bands without any apparent change in the relative positions of the
major bands. Other common glycosidase such as neuraminidase and O-glycosidase did not produce any apparent change in the
electrophoretic mobility of Sh proteins (not shown), suggesting that
they are only N-glycosidated and that, in contrast to rat
brain potassium channels(36) , Sh Kch of Drosophila lack sialic acid in their carbohydrate chains.
Figure 6: Deglycosylation of Sh proteins. Triton X-100 solubilized head proteins were incubated in the absence (A) or presence (B) of endoglycosidase F/N-glycosidase F (see ``Experimental Procedures'') and analyzed by regular SDS-PAGE and Western blot as in previous experiments.
Multiple potassium channel isoforms can presumably be generated by splicing at the Shaker locus of Drosophila(12, 13, 14, 15) . This has raised the question of whether this theoretical repertoire of Sh Kch variants is in fact expressed and how it is used. With our immunochemical approach we have detected a high number of proteins in different anatomical and developmental preparations. The genetic control with Df flies makes the specificity of the detected proteins undoubtable, whereas the control with the mutant W32 demonstrates that the multiplicity of detected Sh peptides is at least in part generated by alternative splicing although post-translational modifications of Sh Kch may also contribute to the complexity of the electrophoretic pattern. Our results clearly contrast with work previously reported by other laboratories which, in spite of the diversity of cDNAs isolated, have shown single bands in Western blot analysis(19, 20) . This is most probably due to the increase in resolution of the electrophoretic techniques used and/or to a higher affinity of the antisera. As we have shown in this paper, the wide repertoire of Sh Kch isoforms in Drosophila is differentially expressed both as tissue as well as in a developmentally regulated manner. Previous reports using in situ hybridization(33) , polymerase chain reaction(37) , and reporter gene DNA engineering (38) have indicated that Shaker transcripts are differentially expressed in some Drosophila tissues. In other organisms where neuronal electrical recording is easier, sharp changes in the electrical properties of neurons take place throughout development together with abrupt changes in the densities and kinetics of potassium currents (see (39) for a recent review). In the same way, two Shaker potassium type A currents with different electrical properties were recorded during the differentiation of Drosophila myotubes showing a progressive replacement of an early type A current by a late mature one(34) . Therefore, our results demonstrating changes in the expression of Sh Kch isoforms during development probably reflect modifications in potassium conductance and, as a result, in the neuronal electrical properties. Further work with isoform specific antibodies will be necessary to identify the Sh Kch variants expressed in different tissues at developmentally defined stages.
Together with the regular
full-length Sh transcripts previously described, the isolation of short
Sh cDNAs that would theoretically produce truncated channel subunits
has been reported by a few
laboratories(3, 12, 13) . The fact that the
corresponding short proteins were not detected in previous studies (19, 20) has made the actual existence of these
truncated proteins doubtful until now. For the first time some specific
short Sh proteins have been detected in this work. They most probably
correspond to the so-called short Sh mRNAs under various criteria.
First, their size (40-45 kDa) fit nicely with that deduced from
the cDNAs having the first three (S1-S3) out of the six membrane
expanding domains of a potassium channel subunit (3, 13) and the possibility of being glycosylated at
the S1-S2 loop(5, 12, 13) . Second, they are
detected by our antiserum -ShHX1 which has been raised against a
recombinant peptide which contains a cytoplasmic stretch common to all
Sh isoforms, the S1 segment and the whole S1-S2 loop. Third, most short
Sh peptides are eliminated by the mutation W32. Thus, our results are
the first evidence for the existence of native truncated Sh proteins
and raise the question of their biological role. Although it has here
been demonstrated that the 45-kDa peptide reaches the plasma membrane,
it is very unlikely that such a truncated Sh protein could drive a
potassium current by itself since it lacks three (S4-S6) out of the six
membrane expanding domains of a voltage-gated potassium channel subunit
and consequently it is missing some of the most important structural
features of a functional channel(6) . However, the short Sh
protein does contain the complete N-terminal and S1 domain where the
structural elements mostly responsible for the assembly of channel
subunits have been localized(40, 41) . Accordingly,
recombinantly produced short potassium channels insert in the membrane
and seem to form heterotetramers with regular large
subunits(42) . Coinjection in Xenopus oocytes of mRNAs
corresponding to large and truncated Sh Kch produce a decrease in the
expressed potassium current (41) suggesting a
dominant-negative role. Transgenic flies carrying a heat inducible gene
encoding a truncated Sh Kch together with regular doses of the wt gene show a Shaker phenotype after heat
shock(43) . Interestingly, a truncated splice variant of the
mouse Kv1-5 potassium channel also shows a dominant-negative
effect (44) . A possible dominant-negative function of the
truncated Sh Kch subunits is a very attractive hypothesis which needs
to be further explored.
Our experiments have shown that the major Sh Kch proteins are evenly distributed in neuronal membranes of different composition and, therefore, strongly suggest a lack of subcellular segregation of major Sh isoforms. Conversely, as mentioned previously, there are reports indicating that certain Sh isoforms are differentially expressed in some areas of the nervous tissue and retina(33, 37, 38) . Nevertheless, our results are not in contradiction with those since extensive work on separation of nervous tissue membranes has demonstrated that fractionation by density centrifugation takes place more as a function of the subcellular nature of membranes than as a result of their cellular origin(29, 45) . In principle, an alternative explanation to our results can be that similar Sh Kch isoforms are detected in membranes at various steps of biosynthesis. However, this is very unlikely since immature channel proteins show a distinct electrophoretic mobility due to differential glycosylation(35, 46) .
The solubilization experiments carried out suggest that about half of the Sh Kch of adult fly brain remains anchored to some submembranous structures and in this way they are resistant to detergents. Other authors have found a similar percentage of solubilized channels in Shaker transfected cells(47) . Interestingly, our detergent resistant pool has the same pattern of Sh isoforms than the solubilized one. This suggests that the structural elements responsible for such an interaction must be within the region common to all Sh Kch subunits. Membrane-cytoskeleton interactions as those found for other ion channels and that seem to play an important role in their distribution and clustering (48, 49) appear as the most likely candidate.
Native Sh Kch proteins from Drosophila have been here demonstrated to be N-glycosylated. The equal shift to lower size of Sh proteins after complete enzymatic deglycosylation leads to the conclusion that at least the major Sh Kch isoforms are glycosylated to a similar extent and probably at the same site(s). However, the contribution of the carbohydrate chains to Sh Kch in different cellular systems (32, 46, 47) range from 16 to 44 kDa which is much greater than the 6 kDa that we have found in native channels. Two consensus sites for N-glycosylation can be found in the sequence of Shaker transcripts at the S1-S2 loop in a region common to all splice variants (5, 12, 13) and both are modified in Shaker channels expressed in Xenopus oocytes (32) and mammalian cells(46) . Unfortunately, with the present data we cannot determine whether one or both sites are glycosylated in the native Sh Kch.
We trust that the immunochemical approach used here can be generally applied to other channel molecules which have already been cloned and present serious difficulties in their standard purification.