Neuron, Vol. 9, 861-871, November, 1992, Copyright 0 1992 by Cell … · 2013. 11. 26. · Figure 1. Enhancement of Expression of K’ Channels by UTRs of a Xenopus P-Clobin Gene

Neuron, Vol. 9, 861-871, November, 1992, Copyright 0 1992 by Cell Press

Subunit Stoichiometry o f a Mammalian K+ Channel Determ ined by Construction o f Mu ltimeric cDNAs

Emily R. Liman, Jan Tytgat, and Peter Hess Department of Cellular and Molecular Physiology and Program in Neuroscience Harvard Medical School Boston, Massachuset ts 02115

Summary

The subunit stoichiometry of the mammal ian K+ channel KVl .l (RCKl) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of ev idence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit fY379K) that expresses only as part of a heteromult imer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.

Introduction

The sequences of Na+ and Ca*+ channels (Noda et al., 1984; Tanabe et al., 1987) contain four internal repeats, each of which bears homology with the sequence of asingleK+channel(Tempeletal. ,1987).Thefunctional K+ channel is, therefore, thought to assemble as a tetramer. It has now been clearly demonstrated by several groups that some K+ channel proteins can form heteromultimers, as ev idenced by the expression of currents with unique propert ies when func- tionally distinct K’channels are coexpressed in Xeno- pus oocytes (Isaacoff et al., 1990; Ruppersberg et al., 1990; Christie et al., 1990) or in a cell line (Ruppersberg et al., 1990). Similar results have been obtained from experiments in which a cDNA construct containing the sequences of two K+ channels in a tandem array was engineered (Isaacoff et al., 1990), suggest ing that the functional K+channel is made up of an even number of subunits. An estimation of the subunit stoichiometry of the Shaker K+ channel has been provided by MacKinnon (1991) from coinjection experiments. Injection of known ratios of cRNA coding for the wild-type Shaker channel (which is highly sensitive to a scorpion toxin) and for a channel with a mutation that confers toxin insensitivity led to the expression of currents with toxin sensitivities that are best fitted by assuming a tetrameric stoichiometry. The results depend on the assumption that hybrid channels are much more sensitive to the toxin than the channel with all mutant subunits and that the mutation does

not affect the efficiency of channel assembly. Despite the e legance of this experiment, a more direct test of thesubunit stoichiometryof the K+channel would be desirable.

This work descr ibes a determination of the subunit stoichiometry of the delayed rectifier K+ channel, KV1.l (RCKI; Baumann et al., 1988; Christie et al; 1989; Koren et al., 1990), based on the engineer ing of constructs containing 2,3,4, or 5 KV1.l channel subunits l inked together in a single open reading frame. As a means of tagging individual subunits, we generated KV1.l channel subunits with mutations at a residue that was expected to decrease sensitivity of the channel to block by external tetraethylammonium (TEA; MacKinnon and Yellen, 1990; Kavanaugh et al., 1991). These mutations, unlike the equivalent mutations in Shaker, eliminate functional expression of the homo- merit KV1.l channel. However, coexpression of these subunits with wild-type subunits (by means of a di- merit construct) reveals that they can contribute to heteromeric channels, as ev idenced by a decreased sensitivity to TEA of the expressed currents. W e find that tetrameric constructs containing 1 or 2 mutant subunits lead to the expression of currents that have altered sensitivities to TEA and that are well fitted by a single site binding isotherm, with a unique value of the concentrat ion of half inhibition, K,. Furthermore, we show that constructs that contain 3 KV1.l subunits l inked together (trimers) can direct the expression of channels, but that these can be dist inguished from channels expressed by a tetrameric construct by their ability to incorporate free monomer TEA-insensitive subunits. This constitutes strong evidence that the mammal ian K+ channel is a tetramer and that subunit stoichiometry and assembly can be controlled in a construct that links together the coding sequences of multiple K+ channel subunits.

Results

Expression Vector To increase expression in Xenopus oocytes of the cDNAs descr ibed in this paper, we constructed avec- tor, pGEMHE, that contained 3’ and 5’ untranslated regions (UTRs) of a Xenopus 8-globin gene (Kreig and Melton, 1984), as shown in Figure 1. Injection of in vitro transcribed cRNA for the KV1.l channel f lanked bythese untranslated sequences resulted in very high expression of K+ channels (Figure IB). K+ currents were detected as little as 10 hr after injection, and after 4 days, currents evoked by steps to a test potential of 0 mV averaged 286 + 35 uA (mean f SEM, n = 13; assessed by extrapolation from currents obtained in 100 mM TEA). This was a dramatic enhancement in the magni tude of the currents when compared with the value of 1.5 + 0.18 uA (n = 16) without the flanking sequences. A IO-fold dilution of the cRNA resulted in

Neuron 862

A

Figure 1. Enhancement of Expression of K’ Channels by UTRs of a Xenopus P-Clobin Gene

(A) Expression vector pGEMHE, constructed as detailed in Experi- mental Procedures. 3’and 5’UTRs from a Xenopus P-globin gene (Kreig and Melton, 1984) flank a polylinker with five restriction enzyme sites. (B) Magnitude of currents in oocytes injected with cRNA transcribed from plasmids that contained the KV1.l channel with or without flanking Xenopus UTRs. Magnitudes of currents for depolarizations to0 mV from a hyperpolarizing holding potential (usually -80 mV) were recorded 4 days after injection. The con- structthatcontainedtheUTRused inthisexperimentwasKVl.l- PSP64T, and the cRNAwas injected at a IO-fold dilution, as injection of the undiluted cRNA killed the oocytes. (C) Effect of the 3’ UTR on the level of expression of KV1.l. Cur- rents were recorded by depolarization to 0 mV at 1 day after injection. The plasmid used for these experiments was pGEMHE containing a wild-type tetramer insert (detailed in Experimental Procedures)and was linearized with Hindlll or Pstl fortranscrip tion. The enzymes cut before or after the 3’ UTRs, respectively. cRNA was injected upon 200-fold dilution of the transcription mix.

only a small reduction in the levels of expression, with a mean current magnitude of 186.2 f 37.9 PA (n = 12), suggesting that the availability of transcript was no longer the rate-limiting step in channel synthesis. Flanking the coding sequence of a second K+channel, KV1.5 (KVI, Swanson et al., 1990; RMK2, Matsubara et al., 1991) with the 3’ and 5’ UTRs of the Xenopus @-globin gene also led to an enhancement of expression by several hundred-fold (E. R. Land P. H., unpub- lished data). These results demonstrate the potency of UTRs of a Xenopus P-globin gene in enhancing the expression of K+ channels in Xenopus oocytes.

Theroleofthe3’untranslated SequenceoftheXeno- pus P-globin gene in enhancing expression was examined by comparing the expression of transcripts that contained this region with the expression of transcripts that lacked it (generated by linearization of the construct with Hindlll). The mean magnitude of the currents was Qfold higher when the transcript contained the 3’ UTR, as seen in Figure IC for one set of oocytes injected with low concentrations of the transcripts (200-fold dilution of the cRNA) and recorded 1 day after injection. A similar enhancement of expression by the 3’ UTR was seen 3 days after injection for the same set of oocytes (7.0 + 0.63 PA, n = 8 versus 1.23 PA, n = 2). This finding is consistent with previous reports on the role of the 3’ UTR of this

KV

Figure 2. Mutations That Alter TEA Sensitivity of ShB and KV1.5 Channel

(A) Sequence alignment of a portion of the pore regions (SSI- SS2) of KV1.l, KV1.5, and ShB. Mutations generated in KV1.l and KV1.5 are shown below (KV1.l) or above (KV1.5) the corresponding sequence. K, values for currents by external TEA are shown beside the sequences. K, was determined as detailed in Experi- mental Procedures and below; values shown are means for more than 4 oocytes. N.E. indicates that no voltage-dependent out- ward currents of >50 nA were observed in at least two batches of oocytes with at least 5 oocytes per batch. Data from mutations in ShB are as in Mackinnon and Yellen (1990), except wild type, which is as in Heginbotham and MacKinnon (1992). (B) TEA inhibition curves for wild-type KV1.5 channels (closed symbols, data from Matsubara et al., 1991) and KV1.5 channels with the mutation R476Y (open symbols). Percent current remaining (I(TEA)/I(Control)) was calculated from the current at each concentration divided by the current in presence of zero TEA. Points are mean for 3-4 oocytes, and bars indicate t SEM. Each data set (single oocyte) was fitted by a least-square minimization of the data (see Experimental Procedures) IITEA]/lcontrol = l/(1 + rEA]/K,). Fits shown are with the mean K, values, which were 0.40 and 600 mM for R476Y and wild type, respectively.

K’ Channel Multimers 863

gene (Kreig and Melton, 1984; Galili et al., 1988) and has important implications for the expression of the multimeric constructs d iscussed below.

Nonexpressing Subunits Are Rescued in Dimers As a marker for the expression of individual subunits, we generated KV1.l channels with mutations at a site (MacKinnon and Yellen, 1990; Kavanaugh et al., 1991) that was expected to alter the sensitivity of the channel to external TEA. Figure 2A shows a sequence alignment of a port ion of the pore regions of the KV1.l, KV1.5 and Shaker B (ShB) channels. Mutations of the threonine (T) residue at position 449 in Shakerto arginine (R) or lysine (K) has been reported to lead to alterations in the sensitivity of the currents to external TEA (MacKinnon and Yellen, 1990), as indicated in Fig- ure 2. Mutation of the equivalent residue in KV1.5 from arginine (R) to tyrosine (Y) has a similar effect (Figures 2A and 2B), where the K, for TEA block is decreased from >I00 mM for the wild-type channel to 0.4 f 0.01 mM (mean + SEM, n = 3) for the mutant. These dramatic effects provide a convenient method for tagging the expression of particular subunits. Mu- tations of the equivalent residue in KV1.l, however, from tyrosine (Y) to arginine (R), lysine (K), or threonine (T) did not lead to the expression of functional channels, as assessed by test depolarizations to 0 mV from holding potentials of -100 to -150 mV. Subclon- ing these mutants into the pGEMHE vector also did not lead to the expression of currents. This indicates that our inability to observe expression of these mu-

tants is unlikely to be due to a decrease in the level of expression.

W e tested the possibility that mutant channel subunits which did not express as homomult imers could express when l inked in a dimeric construct with a wild-type subunit. A dimer was constructed such that an intact carboxyl terminus of the first channel subunit was l inked to the amino terminus of the second subunit, with the addit ion of 17 new amino acids (Fig- ure 3A). Neither this linker, nor a similar linker used for the construction of larger multimers, had any dis- cernable effect on two measures of channel function- ing, gating (Tytgat and Hess, 1992) and permeat ion (E. R. L. and P. H., unpubl ished data). Three different dimeric constructs containing 1 of the 3 nonexpressing subunits, Y379K, Y379R, or Y379T, l inked as the second subunit to a wild-type subunit all led to the expression of large K+currents. The K, for external TEA of these currents was significantly higher than that for the wild type, as seen in Figures 3B and 3C. The higher sensitivity to TEA of the Wt-Y379T dimer compared with the Wt-Y379R and Wt-Y379K dimers is consistent with the K, values for the respective homomeric K+ Shaker channels (Figure 2). This demonstrates that all 3 mutant subunits are capable of contributing to heteromult imers and in doing so cause an alteration in the TEA sensitivity of the resulting channels.

Multimeric Channels and Subunit Stoichiometry To investigate the subunit stoichiometry of the KV1.l channel, cDNAs were engineered by the consecut ive

A

Internal

0.01 0.1

[TEA], i! 10 100

0.0 wt T R K

Figure 3. Dose-Response Curves for Wild- Type Channels and Tandem Constructs

(A) Pictorial representation of the tandem polypeptide. Note that the linker between the 2 subunits is formed from the carboxyl terminus of the first channel l inked to the amino terminus of the second channel. A hypothetical view looking down on a tetra- merit channel formed from 2 dimers in the arrangement ABAB is shown to the right. Since the linker was very long, it is likely that dimers also assembled in an AABB orientation. (B) TEA inhibition curves from tandem construct containing the sequence of a wild- type KV1.l channel fol lowed by the sequence of a KV1.l channel with the mutation Y379T (Wt-T) (open symbols) or Y379R fWt-R) (closed diamonds). Points are mean f SEM for wild-type (Wt) channels (n = 3) and are measurements from single oocytes for the tandem constructs. Solid lines show least-square fits to the data of the equat ion in Figure 1 and correspond to K, values of 0.25 m M (wild type), 2.5 m M (Wt-Y379D, and 9.5 m M fWt-Y379R). (C) Percent current remaining in IO m M TEA for wild-type channels and three tan- dems; a single letter indicates the mutant residue in the second channel of the tandem. Bars show mean f SEM, n = 5,1, 7, and 4 oocytes for wild type, T, R, and K, respectively.

Table 1. Sequential Construction of Multimeric Channels

Construct Vector Insert

Trimer Tri Wt-Y379R-Wt Y2R

Tetramers Tet Wt-Wt-Wt-Y379R Y4R Wt-Y379R-Wt-Wt Y2R Wt-Y379R-Y379R-Wt Y2,3R Y379R-Y379R-Y379R-Wt Y1,2,3R

Pentamers Pent Wt-Wt-Wt-Wt-Y379R Y5R Wt-Y379R-Wt-Wt-Wt Y2R

Di-Wt (C)

Di-Wt-Y379R (D) Tri-Wt (C, D) Tri-Y2R (C, D) Tet-Y2,3R (B, C, D)

Di-Wt-Y379R (E) Di-Wt (E)

Di-Wt-Y379R (A, B)

Tri-Wt (A, B, C) Di-Wt-Y379R (A, B) Di-Wt-Y379R (A, B) Y379R (A)

Tet-Wt (A, 6, C, D) Tet-Y2R (A, B, C, D)

The final construct is given in the first column. The second column shows the abbreviation for the construct. Letters indicate the amino acid substitution tyrosine to arginine and numbers indicate the position in the multimer of the channel that carried the mutation. The last two columns show constructs that were used in generating each new construct, with the channel that they contributed to the new construct (A refers to the first channel) shown in parentheses.

linkingofthecodingsequencesof2-5KVl.l subunits. Table 1 shows the “parent”constructs that were used to generate each new construct, with the subunits that they contributed indicated. All constructs were subcloned into the pCEMHE piasmid, to enhance expression and to select for expression of full-length transcripts (which contain the 3’ UTR). Correct clones were identified by restriction digests.

Tetrameric cDNAs containing a single Y379R subunit as the second or fourth subunit in the coding sequence were constucted. The expectation was that if the constructs generated tetrameric polypeptide chains and if the K+channel assembled as a tetramer, then these constructs should lead to the expression of a single, uniform population of channels, each containing 3wild-typeand 1 mutant subunit. Such a population of channels should have a sensitivity to TEA that is well fitted by a single I:1 binding curve with a K, value that differs from the wild-type value. Figure 4A

shows the dose-response curves obtained from the two tetrameric constructs as well as a third tetrameric construct that contained all wild-type subunits. The inhibition curves for the constructs that contain a single mutant subunit are significantly shifted from that of the wild-type tetramer. The good fit to a single inhibition constant can be seen by the solid lines, which represent the expected sensitivity for a single binding site with Ki = 1.6. The K, values for the two tetrameric constructs containing the same mutant subunit in different positions are not significantly different (p < 0.05, Student’s t test), which is good evidence that full-length polypeptides are being generated, as spec- ified.

To test the uniqueness of this phenotype, we generated pentameric constructs with a single mutant subunit in the second or last position. Dose-response curves obtained from either construct, shown in Fig- ure 4B, are also significantly different from wild-type

Figure4. TEA Sensitivity of Currents Ex- pressed by Tetrameric and Pentameric Constructs with a Single Y379R Subunit

1.0 (A) Block by external TEA of currents ex-

Wt-Y379R-WtGWt t-Y379R-Wt-Wt-Wt pressed by tetrameric constructs in which

WtbWtbWtGY379R 08 a KV1.l channel with the mutation Y379R is 52 either the second (closed symbols) or the 2 Z 06

fourth (open symbols) subunit in the se-

s quence.Thedose-responsecurvefortetra-

=: h

mer containing 4 wild-type channels is

4 0.4 shown for comparison. Symbols show 2 v Wt-Wt-Wt-Wt-Y379R

mean + SEM (when larger than symbol),

0.2 02 with n = 4 oocytes for each data point. Solid lines show the mean fit from least- square fits of each data set (1 oocyte) and

0.0 correspond to K, values of 0.25 and 1.6 mM. 0 01 01

[TEA]. md

10 0.01 01

[TEA]. III; 10 (B) TEA sensitivity of currents expressed by

pentameric constructs with a single mutant subunit (Y379R) in the second (closed sym-

bols) or fifth (open symbols) position (n = 4 oocytes). Dashed lines correspond to fits to wild-type tetramer or tetramer with single mutant subunit from A. Solid lines show fits to data assuming a mixed population of channels with the two K, values (0.25 and 1.6 mM) in the proportions 3:2 (60% wild type) and I:3 (25% wild type). Prediction for random assembly is I:4 (20% wild type). A good fit to the data assuming a single population of channels could not be obtained.


curve. More importantly, in contrast to the results with the tetramer, the inhibition curves for the pentamers containing mutant subunits at different posi- t ions are different from each other. This result is not expected if the channel is a pentamer, or if random subunit mixing is occurring. It is, however, consistent with the assembly of a tetrameric channel from a pentameric construct. The fitted curves in Figure 49 correspond to the sum of inhibition curves for two separate current components, one with the wild-type Ki and one with the Ki for a tetramer containing a single mutant subunit, as determined above. If all 5 subunits have an equal chance of contributing to channel formation, then 20% of the channels should contain all wild-type subunits (l/5 chance that the mutant channel will be excluded) and 80% should contain a single mutant subunit. The relative proport ions of the two populat ions of channels can be estimated from the proport ion of thetwo Ki values that fit the data, assuming that the channels have similar conductances and kinetics. Both these assumptions are likely to be true (data not shown). The currents expressed by the construct with a mutant subunit in the second position conform well to this prediction (fit shown is with 25% wild-type channels), whereas those expressed by the construct with a mutant channel in the fifth position (fit shown is with 60% wild-type channels) deviate significantly from this prediction. This indicates that position in a pentameric construct is important in determining the probability that a subunit will contribute to the functional channel.

Coinjection of Tetrameric and Trimeric Constructs with Y379K An alternative test of the subunit stoichiometry of the KVI.l K+ channel was based on a compar ison of the expression of constructs containing 4 wild-type KVI .I channels or 3 wild-type KV1.l channels. Contrary to expectation, both constructs led to the expression of large K+ currents. Expression of the trimer in one experiment gave a mean current ampli tude of 0.39 f 0.01 PA (n = 4) at 0 mV; this was lower than the expression of the tetramer, which in a different experiment gave a mean current ampli tude of 7.0 f 0.63 WA at 0 mV (n = 8) for a similar concentrat ion of cRNA.

The functional expression of trimericas well as tetra- merit constructs suggested that, in the absence of a full complement of subunits on a single polypeptide, functional K+ channels could be formed by subunits that were part of separate polypeptides. The availability of a mutant channel, Y379K, that expressed only in heteromult imers and that altered the pharmacologi- cal profi leofthecurrent al lowed ustotestthis hypothesis. If a functional channel is generated by subunits on the same polypeptide, then coexpression with the mutant subunit should have no effect on the sensitivity of the expressed currents to TEA. This is because, at modest levels of expression, the subunits on the same polypeptide will be at essentially infinite concentrations relativetotheconcentration ofcfree mono-

meric subunits. In contrast, if the polypeptide contained fewer than the number of subunits necessary to form a functional channel, coexpression with the mutant channel should result in the formation of heteromultimers. Figure 5A shows the currents that we recorded from oocytes coinjected with the trimer and the mutant subunit or the tetramer and the mutant subunit. Dose-response curves, shown in Figure 59, for the coinjection of the tetramer with Y379K are in- distinguishable from those obtained from the tetramer alone, whereas those from the coinjection of the trimer with Y379K are significantly shifted compared with those from the trimer alone. The trimer alone, tetramer alone, and wild-type monomer are all equally sensitive to TEA, as expected. Similar results were obtained in two additional experiments in which trimer cRNA was coinjected with Y379K cRNA. In these experiments 3.3 mM TEA blocked 54% f 2% of the current (n = 5) for coinjection of a low concentrat ion of trimer cRNA (IOOO-fold dilution) and 62% + 1% (n = 2) for a higher concentrat ion of trimer cRNA @O-fold dilution) compared with -93% block of the wild-type channels.

The absence of any detectable heteromult imer formation when the tetramer is coinjected with the mutant channels allows us to conclude that the construct is generat ing full-length multisubunit polypept ides and that the number of subunits in this polypeptide is precisely the number of subunits that form a functional channel, or is an even multiple of the number that form a functional channel (i.e., 2 subunits make a functional channel). The observat ion that the trimer does readily form heteromult imers demonstrates that the mutant subunits in the above experiment were competent to contribute to heteromultimers. Further- more, it demonstrates that the functional channel is not a trimer, and it is consistent with a tetrameric structure of the K+ channel. Indeed, the dose- response curve from the coinjected trimer is well fitted by the sum of two Ki values, one set to the wild- type value and the other chosen to provide the best fit to eye to the data. The value of the second Ki (3 mM) is slightly higherthan thatof thetetramerwithasingle Y379R mutant, consistent with our observat ion that the Wt-Y379K dimer is less sensitive to TEA than the Wt-Y379R dimer (Figure 3C) and that mutant Shaker K+ channels with a lysine at the equivalent position are less sensitive to TEA than mutants with an arginine at this position (MacKinnon and Yellen, 1990).

The trimeric construct, when expressed alone, thus forms functional channels by donat ions from at least 2 polypept ides to a single channel. This assembly is likely to be fairly inefficient, and one would predict that expression could be enhanced bythe coinjection of free subunits. This is indeed the case, as seen in Figure 5C. Whereas coinjection of mutant subunits had no significant effect on the magni tude of currents expressed by tetrameric channels (p > 0.5, Student’s t test), coinjection with the trimeric construct led to a more than 4fold,enhancement inthe magni tudes of

Neuron 866

A Wild-type KV1.1 Tetramer

/- 00 0.1

- :

p5 uA 20 ms

i- 0 mv -1

Co-injection with Y379K Tet + Y379K

Tn + Y3’79K

/---- ---- o y----- 0 I

Kk--- I --PI-

T-

001 01 I I 0

[TEA]. mh4

Figure 5. Channels Expressed by Trimeric Constructs, but Not Tetrameric Con- structs, Can Form Heteromultimers

(A) Currents evoked by step depolarization to 0 mV in the presence of varying concentrations of external TEA. The TEA concentration (millimolar) is indicated beside the corresponding trace. Oocytes were injected with cRNA from a tetramer containing 4 wild-type channels or from a tri- mercontaining3wild-typechannels(traces not shown), or they were coinjected with cRNA from the Y379K mutant and the wild- type tetramer or wild-type trimer. cRNA was diluted from a standard transcription volume by40-fold for the trimerand by400- fold for the tetramer. The same transcription of Y379K cRNA at the same concentration (200-fold dilution) was used for both coinjections. (B) TEA sensitivity of the wild-type tetramer (open diamonds), wild-type trimer (open circles), and wild-type tetramer coinjected with Y379K (closed diamonds) fitted with a solid line corresponding to a K, of 0.25 mM. TEA sensitivity of the wild-type trimer coin- jetted with Y379K (closed circles) fitted with a solid line corresponding to the prediction for two populations of channels with K, values of 0.25 and 3 mM in the pro- portion 1:3. Data are from same injections as in (A).

(C) Enhancement of current amplitude in oocytes coinjected with the Y379K channel. Trimer cRNA injected alone (200-fold dilution) or coinjected with Y379K (200-fold dilution); the concentration of trimer cRNA was kept constant. Amplitude of currents evoked in a step depolarization from -100 mV to 0 mV, normalized by the mean amplitude of currents in oocyte injected with trimer alone. Tetramer injected alone (400-fold dilution) or coinjected with Y379K (200-fold dilution). Same normalization procedure. Bars show mean + SEM based on recordings from 4-8 oocytes.

the currents expressed. Similar enhancements in the magnitudes of the currents were seen in two other experiments, with either a IO-fold higher concentration of trimer cRNA (4.0 + 1.5-fold enhancement, n = 5 control and n = 6 coinjected oocytes) or a IO-fold lower concentration of cRNA (4.2 + 0.7-fold enhancement, n = 6 control and n = 6 coinjected oocyte), in two different batches of oocytes. This result is in nice agreement with the dose-response curves, which show that when the trimer is coinjected with Y379K, approximately25% of the channels are wild-type. This suggests that there is a pool of polypeptides that do not form functional channels when the trimer is expressed alone. That is, if all the trimeric peptides contributed to functional channels (i.e., trimers dimerize), then we should observe at most a doubling in the size of the currents. Similarly, these results make it unlikely that the functional channel is a dimer, since in that case also, at most a doubling in the size of the currents is predicted. The size of the pool of unassem- bled, or partially assembled, channels cannot be estimated from these experiments because the concentration of mutant cRNA was kept deliberately low (in order to prevent any competition for translation ma- chinery that might have diminished expression). It is

possible, therefore, that higher concentrations of mutant cRNA would have led to even more dramatic enhancement of the currents.

Tetrameric Constructs with Varying Numbers of Mutant Subunits To investigate further the phenotypes of the tetra-

merit constructs, 2 additional tetrameric constructs were generated, with 2 Y379R subunits (as the second and third subunits) and with 3 Y379R subunits (as the first, second and third subunits). Since the mutant channel did not express alone, the fifth type of tetramer, one with 4 mutant subunits, was not generated. Figure 6A shows currents recorded from oocytes injected with cRNA for channels with 0, 1, or 2 mutant subunits, demonstrating the incremental decrease in sensitivity to TEA seen among the 3 channels. Dose- response curves for channels with 0, 1,2, or 3 mutant subunits are shown in Figure 66. The tetramer with 2 mutant subunits is well fitted by a K, = 10.5 mM (n = 4), which is not significantly different from the K, of the Wt-Y379R dimer and is significantly greater than that of the tetramer with a single mutant subunit. The tetramer with 3 Y379R channels expressed extremely poorly (generally less than 100 nA, 2 independent


(A) Currents evoked by step depolarizations to 0 mV with varying concentrations of TEA in the bath, shown to the right of the traces. Constructs contained 4 wild-type

0 Wt Tetrarner channels, 3 wild-type channels, and 1 Y379R channel in the second position, or 2 wild-type and 2 Y379R channels in the second and third positions. (6) TEA sensitivity of tetrameric constructs, as above, with 0 (closed circles), 1 (open circles), 2 (closed diamonds), or 3 (open dia-

, ’ mends) mutant subunits. Points are mean + SEM for 3-4 oocytes. More data point could not be readilyobtained from thecon- struct with 3 mutant channels because of extremely poor expression. (C) Semilog plot of K, values of channels from tetrameric or dimeric constructs, as a function ofthe number of mutant channels in the construct. For the dimeric con-

_I 01 1 10

structs, the number of mutant channels 100 0 1 2 3 4 was assumed to be twice the number in

[TEA]. rnM No of mutant subunIts the construct. Data points are mean f SEM from oocytes in which complete dose-response curves had been obtained (except the tetramer with 3 mutant subunits). Zero

mutant subunits: Wild-type tetramer (open circles; n = 2) and wild-type monomer (closed circles; n = 3); 1 mutant subunit: tetramer with Y379R in the second position (closed symbols; n = 4) and in the fourth position (open symbols; n = 4); 2 mutant subunits: tetramer with Y379R in the second and third positions (closed circles; n = 4), dimer Wt-Y379R (open circles; n = 4), dimer Wt-Y379K (closed squares; n = 3), and dimer Wt-Y379T (closed diamonds; n = 1); 3 mutant subunits: tetramer with Y379R in the second, third, and fourth positions (closed circles; n = 3). Straight lines were fitted by eye to the data.

clones tested), but measurements from the oocytes with the largest currents show that this channel is even less sensitive to TEA (n = 3). These currents had activation curves and a Rb+ to K+ selectivity that was not noticeably different from that of the wild type. The existence of five (including the nonexpressing homomultimer) pharmacologically distinct channel types is further evidence that the K+ channel is com- posed of at least 4 subunits.

The equal spacing on a logarithmic scale of the K, values of dimeric channels with combinations of 2 different subunits has been used to argue that TEA is energetically stabilized in the pore of the channel by additive contributions from all subunits (Heginbo- tham and MacKinnon, 1992). If the same principle ap- plies here, then for a tetrameric construct, we can express the contributions to the free energy of binding of TEA from 2 different subunits, a and b, with the stoichiometries of n and 4- n, where n varies between 0 and 4, as

AGhybr,d = (n/4) X AC”, + (1 - n/4) x AGob.

It follows that

(1)

Ln(Khybr,d) = (114) X [LnfK,) - Ln(Kb)] X n + Ln(Kb), (2)

where AGo, is the free energy of binding of TEA to a channel with all a subunits, and K, is the respective

K, for block of this channel (see Heginbotham and MacKinnon, 1992). From this last equation it can be readily seen that the logarithm of the Ki of a hybrid tetrameric channel should vary linearlywith the number of mutant subunits. Our results are in nice agreement with this prediction, as demonstrated in Figure 6C, which plots the mean K, values for all the tetrameric and dimeric channels on a logarithmic scale as a function of the number of mutant subunits in the channel. The good fit of the data with the mutation Y379R to a straight line allows us to predict the Ki of the homomeric Y379R to be - 400 mM. Similarly, the K, of the homomeric Y379T channel can be estimated to be -24 mM, and the homomeric Y379K channel can be estimated to be -1700 mM. These values are in good agreement for the K, values of Shaker K+ channels with the same residues at the equivalent position (MacKinnon and Yellen, 1990). Furthermore, the calculated K, of 2.3 mM for the tetramer with a single Y379K subunit justifies the value used in the fit to the data from the coinjection experiments above.

Discussion

The results of our experiments are summarized pictorially in Figure 7. The observation that channels expressed by a tetrameric construct do not incorporate free monomer subunits is strong evidence that the

1 mutant subunit

Tetramer 1 Ki No effect of positlon

Pentamer 2 K,s Proportions depend on position of mutant subunlts

Co-injection with mutants subunit Figure 7. Summary of Experiments

K’ channels are represented pictorially. The view is looking down on the channel.

@

Mutant subunits are represented in gray,

0, I__

and linkers are represented by loops. Re-

0

suits from experiments in which constructs contained a single mutant subunit are

Tetramer wt K, No enhancement

shown on the left. While the position of the mutant subunit does not influence the

Trlmer K, i wt Enhancement

functional channel formed by tetramers, it may influence the functional channel formed by pentamers. For example, the last subunit may be more likely to be excluded because it is lessconstrained than the middle subunits. Results from coinjection of trimeric and tetrameric constructs with mutant subunits are shown on the right. Note that 2 trimeric polypeptides that have irreversibly assembled with themselves cannot form a functional channel, but will readily form functional channels when combined with singlesubunits. Monomers may also aggregate into such nonfunc- tional trimers.

KV1.l K+ channel is a tetramer. It is also evidence that our construct does not generate a significant amount of truncated polypeptides, as might occur if thecRNA has been degraded, or if translation does not go to completion. In this case, we would have expected to see evidence of heteromultimer formation (i.e., a decrease in sensitivity to TEA) regardless of the number of subunits encoded by the cDNA. The observation that the sensitivities to external TEA of tetrameric channels containing a single TEA-insensitive subunit are well fitted by a single K,, irrespective of the position of the subunit in the construct, is further evidence that full-length polypeptides are generated by the tetrameric constructs and that the KC channel is a tetramer. Had these constructs somehow resulted in the expression of unlinked subunits, then we would have expected to obtain dose-response data that could not be fitted by a single I:1 binding curve, as previously demonstrated in experiments in which monomers that differ in sensitivitytoTEAwerecoinjected (Christie et al., 1990; Matsubara et al., 1991). If translation of these tetramers was prematurely terminated, the mutant subunits, which were positioned at the end of the construct, would not have been translated as effectively as the wild-type subunits, and we would have observed a component of the current that was wild type with respect to TEA block.

A surprising result of these experiments is that cDNAs encoding 3 or 5 K+ channel subunits linked together led to the expression of currents. The expression of currents by the trimeric construct means that subunits encoded on separate polypeptide chains are contributing to the functional K+ channel. Channels could be formed by equal contributions from two trimers (2 subunits each), by unequal contributions

(Figure 7), or by contributions from more than two trimers. A pool of polypeptides that assemble as non- functional trimeric oligomers which cannot hetero- polymerize with each other because they cannot present a full contact surface is probably also generated (see Figure 7). The relative size of this pool is probably quite underestimated in our coinjections experiments, as only low concentrations of mutant subunits were coinjected. A similar phenomenon may exist in native preparations in which channels are assembled from monomer subunits. If assembly is essentially ir- reversible at each step, then partially assembled tri- merit channels could be present at high concentrations. These channels could give rise to gating current as well as boosting, through heteropolymerization, the expression of newly synthesized monomer subunits. An understanding of the assembly of K+ channels could thus have important biological and bio- physical consequences.

The pharmacology of channels expressed by pentameric constructs may also provide information on subunit assembly. The observation that a subunit encoded last in the pentamer contributes to -40% of the functional K+ channels rules out a mechanism whereby irreversibleassemblyof each subunitoccurs immediately upon translation. The contribution of the last subunit is, however, significantly less than the valueof80X predicted if all subunitsareequallylikely to contribute to the functional channel. Several expla- nations for this observation are possible. From the results of the experiments with tetramers, it appears unlikely that this is the result of RNA degradation or partial translation. A comparison with the results from the pentamer with a mutant subunit in the second position, in which the mutant subunit contributes to


-75% of the functional channels, indicates that this is not due to an effect of the mutation on the assembly ofthesubunit.Anotherpossibilityisthatthisselective exclusion is a result entirely of the fifth channel being linked at only one end to the other subunits and thus less constrained than the middle subunits. In this case we would expect the first subunit to be equally excluded. Since the fifth subunit is excluded >50% of the time, the first subunit would be predicted to be excluded the remaining 50% of the time and the 3 middle subunits would never be excluded. Although the data from the pentamer with a mutant subunit in the second position are not consistent with this hypothesis, this question would be addressed best by a systematic construction of pentamers containing mutant subunits at each position. These results demonstrate the potential usefulness of the generation of pentameric K+ channel polypeptides in studying the relative efficiencies of assembly of different naturally occurring or mutant subunits.

Our results from tetramers that contain from 0 to 3 TEA-insensitive subunits are in extremely good agreement with similar experiments with dimers of Shaker K+ channels (Heginbotham and MacKinnon, 1992) and of KV1.l channels (Kavanaugh et al., 1992). Those experiments have convincingly demonstrated that external TEA does not interact independently with each subunit, and from dimeric constructs of Shaker channels it has been proposed that the TEA ion is energetically stabilized by simultaneous contributions from all the subunits (Heginbotham and Mackinnon, 1992). The same type of energy additivity is seen in the tetramers of KV1.l channels with 0, 1, or 2 TEA-insensitive subunits. Thus, although these TEA-insensitive subunits do not form functional ho- momultimers, the heteromultimers that they form must have a binding site for TEA similar to that of the Shaker K+ channel.

ByflankingthecodingsequencesoftheKV1.1 channel with UTRs from a Xenopus P-globin gene, we have found that expression is dramatically enhanced. These regions have previously been shown to enhance expression of other proteins (Kreig and Melton, 1984; Galili et al., 1988; Falcone and Andrews, 1991). The over IOO-fold enhancement that we observe for the expression of K+ channels expressed in Xenopus oocytes is similar to the level of enhancement seen for some other proteins upon addition of the flanking 5 Xenopus P-globin UTR, as assessed in a reticulocyte lysate system (Falcone and Andrews, 1991). The rela- tively smaller effect of the 3’ UTR, which enhanced expression only by 4 to 5-fold, is similar to that reported by others for a maize zein transcript (Galili et al., 1988). This expression construct may therefore be widely useful for the efficient expression of exoge- nous proteins in Xenopus oocytes.

In summary, we have demonstrated that constructs linking the coding sequences for multiple K+channel subunits can direct the expression of K+ currents. These constructs have provided strong evidence that

the K’channel is a tetramer, and they may prove useful in the exploration of subunit assembly and in the assessment of the extent and nature of subunit inter- actions.

Experimental Procedures

High Expression Plasmid The KV1.l and KV1.5 channels were subcloned into the PSP64T vector (gift of D. Melton), which contains 5’ and 3’ UTRs from a Xenopus P-globin gene (Kreig and Melton, 1984). A single Bglll restriction site in thevector wascut and made blunt with Klenow. The KV1.l insert was generated by digestion with EcoRI and Hind- III and was made blunt with Klenow. The KV1.5 insert was generated by partial digestion with EcoRI, complete digestion with Hindlll,and bluntingwith Klenow. Both insertswereligated into the PSP64T construct, and clones containing inserts in the right orientation were identified by restriction digest (KVl.l-PSP64T and KV1.5-PSP64T). The ligation destroyed the EcoRI, Hindlll, and Bglll sites. cDNA was linearized with BamHI, and cRNA was transcribed with SP6 polymerase.

The KV1.l + P-globin UTR and KV1.5 + P-globin UTR were subcloned into the pSelect vector (Promega Corp.) such that sense RNA would be read off by T7 polymerase. KVl.l-PSP64T was cut with Hindlll, blunted, and then cut with Pstl to yield a ‘2 kb fragment containing the KV1.l coding sequence and p-globin UTR. This fragment was ligated to the pSelect vector and cut with Smal and Pstl. Correct clones were identified by restriction digest (KVl.l-HE-pSelect). The KV1.5 + P-globin UTR-pSelect construct (KVl.S-HE-pSelect) was generated in a similar manner except that Sacl was used in lieu of Pstl. cRNA was transcribed by T7 polymerase.

Ageneral high expression construct (pGEMHE)was made from the KVl.l-HE-pSelect construct. EcoRl and Hindlll sites that flanked the insert were destroyed by digestion with each of the enzymes, blunting, and religation. Single-stranded DNA was made from this construct, and a mutation was introduced that added a sequence of restriction sites to the 3’end of the channel coding sequence. The first site in the new polylinker, Xmal, is also found at the 5’ end of the channel coding sequence, and thus digestion with Xmal lifts out the channel and leaves the P-globin UTRs, now with a good polylinker. This was subcloned into the PCEM-3Z vector, and the final vector is shown in Fig- ure 1.

Mutagenesis The “altered sites” system (Promega Corp.) was used. The coding sequences of KVI .I and KVI .5 were inserted, after digestion with EcoRl and Hindlll (partial for KVl.S), into the pSelect-I vector. KVl.l-HEand KVl.S-HEwereinsertedasdescribedabove. Muta- tions were verified by dideoxy DNA sequencing. For each construct, several independent clones were sequenced and transcribed. The plasmid was linearized with an appropriate enzyme (Pstl or Hindlll) and cRNA was transcribed with T7 polymerase. After ethanol precipitation, the cRNA was resuspended in 20 pl of dH,O.

Dimeric cDNA Construction An EcoRl site at the beginning of the coding sequence of the KV1.l cDNA Wend) was eliminated, and another EcoRl site was introducedat theendof thecodingsequence, just5’toaHindlII site (before the stop codon). Digestion of this construct with EcoRl and Hindlll opened the vector at the end of the coding sequence of the KV1.l channel (the A channel). The second (B) channel was isolated as the 1.6 kb fragment from an EcoRl and Hindlll digest of the pGEMHE construct with a single channel insert, and the two fragments were ligated overnight. The segment that linked the sixth transmembrane domain of the A channel and the first transmembrane domain of the B channel thus consisted of the entire carboxyl terminus of the A channel, the entire amino terminus of channel B, plus 17 new amino acids. This segment, derived from the 5’ UTR of the KV1.l clone of

NfWV3ll 870

the B channel between the EcoRl site and the normal initiation codon, contained the amino acids LHPGLSPGLLPLHPASI. This construction resulted in the elimination in the A channel of a Dral site located at the end of the coding sequence of the channel.

Multimer Construction Trimers Insert: The entire coding sequence of a dimer was isolated as the 3.2 kb fragment from a double digest of the dimer construct with Kpnl and Dral (which generates a blunt end); thesechannels constituted the A and B channels in the final construct.

Vector: A fragment containing the C channel fol lowed by the vector was generated as follows: A dimer was digested with Xmal, which cuts at the Send of both the A and B channels, and the ends were made blunt by Klenow. After heat inactivation of the Klenow, the DNA was cut with Kpnl and a 4.6 kb fragment containing the 6 channel of the dimer and the vector was isolated. The two fragments were l igated to yield a construct of 7.8 kb. Note that the Xmal site in the C channel was el iminated in the blunt end ligation with the Dral site of the A + B dimer.

The two fragments were l igated overnight. Note that the links between the carboxyl and amino termini of the B and C channels contained the last 15 of the 17 amino acids listed above for the dimer construct. Tetramers and Pentamen Insert: Double digestion of monomers, dimers, trimers, or tetramers with Kpnl and Dral yielded fragments that contained the entirecodingsequenceofthemultimer(i.e.,1,2,3,or4channels) of 1.6, 3.2, 4.8, and 6.4 kb respectively.

Vector: Fragments were generated as above by digestion with Xmal and Kpnl. Since the Xmal site was el iminated in the beginning of the coding sequence of the C (D or E) channel, this digestion generated a fragment that contained all the channel coding sequences after the A channel, fol lowed by the vector.

Expression and Two Microelectrode Recording from Xenopus Oocytes Preparation and injection of oocytes and two microelectrode recording were as described in Koren et al. (1990). Recordings were performed from 10 hr to 5 days after injection at room temperature (- 22OC).

TEA Sensitivity Control bath solution contained 2 m M KCI, 118 m M NaCI, 1 m M MgCI,, 0.3 m M CaCIZ, and IO m M HEPES (pH 7.2). TEA chloride was substituted for equimolar concentrations of NaCl in the test solutions. The ampli tude of the currents at a constant test potential (usually 0 mV) in the solution containing TEA was compared with the control value before and after application of the test solution. Adjustment for drift in the control values was based on the assumption that the drift was consistent throughout the experiment. Experiments in which the currents were larger than 2.5 PA or in which the drift was greater than 10% were not in- c luded in the analysis. Dose-response curves were fitted with a I:1 binding curve by least-square minimization.

Acknowledgments

This work was supported by the USPHS and a Howard Hughes Medical Institute predoctoral fellowship (to E. R. L). 1. T. is a Senior Research Assistant of the N. F. W. 0. (Belgium). P. H. is an established investigator of the American Heart Association. W e thank Drs. B. Bean and R. MacKinnon for critical reading of the manuscript, Dr. M. White for suggesting the use of the Xenopus P-globin untranslated regions, Dr. G. Strichartz for suggesting the construction of pentamers, and Drs. A. Rittenhouse, C. Kuo, and D. Slish and Ms. K. Chua for helpful discussions.

Correspondence should be addressed to E. R. L iman at her present address: Department of Neurobiology, Harvard Medical

School, 220 Longwood Avenue, Boston, Massachusetts 02115. The costs of publication of this article were defrayed in part

by the payment of page charges. This article must therefore be hereby marked “advert isemenf” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received June 16, 1992; revised August 18, 1992.

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Documents

Neuron, Vol. 9, 861-871, November, 1992, Copyright 0 1992 by Cell … · 2013. 11. 26. · Figure 1. Enhancement of Expression of K’ Channels by UTRs of a Xenopus P-Clobin Gene