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J. Biol. Chem., Vol. 279, Issue 2, 1003-1009, January 9, 2004
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From the Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom
Received for publication, July 24, 2003 , and in revised form, October 8, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Molecular cloning studies have identified three closely related genes (SK1, -2, and -3) which code for SK channel subunits in mammalian cells (5–7). In both Xenopus oocytes and mammalian cell lines, expression of the rat homologues of SK2 and SK3 (rSK2 and rSK3 respectively) results in the formation of functional homomeric SK channels. Further, both homomeric rSK2 and homomeric rSK3 channels can be blocked by either apamin or UCL 1848 at concentrations that are similar to those reported for native channels (8). The potencies of both compounds depend on the subunit composition of the channel, with the IC50 for blocking homomeric SK2 channels being 18-fold lower than for SK3.
The behavior of SK1 is different to that of other SK genes. Initial expression studies of the human SK1 gene (hSK1) showed that it can produce functional channels in the Xenopus oocyte expression system and these channels are insensitive to apamin at concentrations of up to 100 nM (5). Subsequent work, however, expressing hSK1 in mammalian cell lines, showed that most cells produce channels that are blocked by apamin with an IC50 of 3–12 nM. A few cells also produce an apamin-insensitive current component (9, 10). More recent oocyte work suggests that, in fact, expression of the human SK1 gene also produces some apamin-sensitive channels in this system (11). The reason for this behavior is unclear and it consequently remains unknown whether SK1 forms apamin-sensitive channels or apamin-insensitive channels, or both, in vivo. Interestingly, the rat SK1 gene differs from its human counterpart because it does not produce functional channels when expressed alone in either oocytes or mammalian cell lines (12).
The overlapping patterns of expression for SK1, SK2 and SK3 within the CNS (5, 13, 14) raise the possibility that heteromeric assembly of SK subunits may occur. That co-assembly may occur is also suggested by the work of Ishii et al. (15) where it was shown that injection of an SK1-SK2 dimer or co-injection of mRNA for hSK1 and rSK2 into Xenopus oocytes resulted in currents with an apamin sensitivity between that of homomeric hSK1 and rSK2. Following this observation we have recently shown that co-transfection of the rat SK1 and SK2 genes in HEK cells produces channels with a novel pharmacology, suggesting that subunits from these genes can also assemble to form functional heteromeric channels (16). Despite the progress being made toward understanding SK1/SK2 interactions, much less is known of the interactions between SK3 and the other SK subunits. Interestingly, however, it has been reported that a fragment of SK3, when transfected into the Jurkat cell line, acts as a dominant negative, suppressing the endogenously expressed SK2 current (17). This finding suggests that SK2 and SK3 may be able to form a heteromeric complex. The principle aim of the present work was to examine, in more detail, the possibility that SK3 interacts with the other SK channel subunits; SK1 and SK2.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Maintenance and Transient Transfection of HEK 293 Cells—HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% v/v fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were plated into 35-mm culture dishes and transfected using LipofectAMINE 2000 (Invitrogen). The protocol for transfection of HEK cells was varied slightly according to the precise aims of the experiment. For studies of the pharmacology of homomeric SK channels we used 1 µg of plasmid. For experiments where hSK1YP or rSK1 were co-expressed with another SK gene we used 2 µg of hSK1YP or rSK1 construct to 1 µg of the other. For co-expression of hSK1 and the change-of-function mutants 1 µg of each was used. For immunohistochemistry cells were transfected with 1 µg of rSK3 alone or with 1 µg of rSK3 together with 4 µg of hSK1 or rSK1. In addition, for all experiments, cells were co-transfected with 1 µg of QBI plasmid DNA (Qbiogene), which expresses GFP, allowing identification of transfected cells. LipofectAMINE 2000 (1 µl/µg of DNA) and plasmid DNA were mixed in Opti-MEM and added to cells plated into 35-mm culture dishes. After incubation overnight the cells were re-plated into 35-mm dishes and used within 2 days for recording. Cells used for immunohistochemistry were plated on 18-mm square glass coverslips.
Immunohistochemistry—Transfected cells plated on glass cover slips were washed three times in phosphate-buffered saline (PBS) before being fixed for 10 min using freshly made 4% w/v paraformaldehyde (in PBS). Cells were washed in PBS again and permeabilized using 100% methanol for 10 min. After a further wash in PBS the cells were then left in an antibody-blocking solution (ABS) consisting of 2% w/v bovine serum albumin and 2% w/v horse serum in PBS for 1 h. The cells were then incubated for 4 h in the primary anti-rSK3 antibody (Chemicon) at a concentration of 0.3 µg/µl. To remove excess primary antibody, the cells were washed three times in PBS containing 0.1% v/v Tween-20 and then incubated in a 1:200 dilution of a Cy3-conjugated goat anti-rabbit secondary antibody (Chemicon) for 1 h. All antibodies were diluted in ABS. Following incubation, cells underwent a final wash in PBS with 0.1% v/v Tween-20. Coverslips were mounted onto slides (previously cleaned with ethanol) using a small drop of antifade mount (Vector Laboratories Inc.). All staining operations were carried out at room temperature (22 °C). Stained cells were viewed with a Leica TCS confocal microscope.
Electrophysiology—Currents were recorded from HEK 293 cells using conventional whole cell voltage clamp methods. The bathing solution contained (in mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, and the pH was adjusted to 7.4 with NaOH. The pipette filling solution contained (in mM): KCl 130, HEPES 10, K2HEDTA 5, and CaCl2 1.2 (free Ca2+ 1 µM). The pH was adjusted to 7.2 with KOH. The free Ca2+ concentration was calculated using stability constants from Martell and Smith (18). The 50 µM (unbuffered) calcium pipette filling solution contained (in mM): KCl 140, HEPES 10, and 0.05 CaCl2 (pH adjusted to 7.2 with KOH). Patch pipettes were fabricated from 1.5 mm o.d. borosilicate glass (Harvard), fire-polished, and coated with Sylgard resin. They had resistances of 2–4 M
when filled with the above solution. Experiments were conducted at room temperature (20–25 °C).
Membrane currents were recorded with either a List EPC7 amplifier using a Digidata 1320A interface and pClamp 8.2 software (Axon Instruments) for acquisition, or a HEKA EPC9 patch clamp amplifier under control of Pulse software. Data were filtered at 1 kHz and digitized at 5 kHz. Acquired current traces were analyzed with either Clampfit 8.2 or HEKA Pulsefit.
Routinely, cells were held at –80 mV and current-voltage relationships generated by applying 100 ms voltage steps to potentials between –120 mV and +40 mV. Since untransfected (wild-type) HEK 293 cells exhibit an outwardly rectifying current at potentials positive to 0 mV the effect of blockers was measured at –20 mV. Similarly, comparisons between currents in cells transfected with or without dominant negative construct were made at this potential.
Data Analysis—The effect of blocking agents was expressed as the current at –20 mV in the presence of blocker as a percentage of that in its absence. The resulting concentration-inhibition curves were fitted by the Hill equation in the form of Equation 1,
 | (Eq. 1) |
Drugs and Reagents—All materials used for tissue culture were obtained from Invitrogen. UCL 1848 (8,14-diaza-1,7-(1, 4)-diquinolinacyclotetradecaphanedium tetratrifluoroacetate) was synthesized in the Department of Chemistry, UCL as previously described (2). Tetraethylammonium chloride (TEA) was purchased from Sigma. HEPES and HEDTA were from Calbiochem. All other reagents were of Analar quality and obtained from Merck or Sigma.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 min washout (Fig. 1, A and C). This cannot be obtained with apamin (10).
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Effect of a Dominant Negative hSK1 Subunit on rSK3 Currents—Next, to examine possible assembly of SK3 with SK1 we used a dominant negative construct of hSK1 (hSK1YP) (Fig. 2). We performed three types of control experiments with this construct. First, we tested hSK1YP co-expression with hSK1 to make sure that it can knock down the wild-type SK1 currents. To do this we compared the size of currents in cells transfected using SK1 alone with those transfected using the same quantity of SK1 and a 2-fold excess of hSK1YP. As expected, the hSK1YP construct substantially reduced the SK1 current (Fig. 2D). Second, to confirm the effectiveness of this construct in co-assembly experiments, we expressed SK1YP with the wild-type rSK2 gene. The human SK1 gene and the rat SK2 gene have been shown to form subunits that co-assemble in oocytes. We therefore expected to see knock down of SK2 current. Again we compared the size of currents in cells transfected using SK2 alone with those of cells transfected using the same quantity of SK2 and a 2-fold excess of SK1YP. As expected, there was a substantial reduction in currents when cells were co-transfected with the dominant negative (Fig. 2, B and D). This result is consistent with the idea that in mammalian cells, as well as in oocytes, SK1 and SK2 can co-assemble (even though the pharmacological properties of hSK1 appear to be quite different in the two expression systems). Interestingly, there was more current remaining than would be predicted if both constructs express equally well, subunits assembled at random and only a single subunit of hSK1YP were sufficient to render SK1/SK2 channels nonfunctional. The reason for this is not clear. It may reflect either a partial recovery of function, preferred stoichiometries of the SK1/SK2 interaction or variations in expression levels. However, to ensure that overexpression of a competing plasmid does not itself cause the apparent reduction in SK currents, we performed a final control experiment testing the effect of hSK1YP on the current in cells transfected with hIK1. As in the previous cases, we used a 2-fold excess of the hSK1YP construct over the hIK1 plasmid and compared the currents against those obtained using hIK1 alone. In these experiments the magnitude of the mean hIK1 current was slightly, but not significantly, reduced as a result of co-transfection with hSK1YP (Fig. 2, C and D). This suggests that competition for expression by a 2-fold excess of the hSK1YP construct does not account for the reduced currents seen when hSK1YP is transfected with wild-type SK1 or SK2. The hSK1YP construct thus seems suitable for assessing subunit co-assembly. Having completed these control experiments, we compared cells transfected with SK3 alone or cells transfected with the same quantity of SK3 and a 2-fold excess of dominant negative. The records shown in Fig. 2, A and D demonstrate that SK3 currents were greatly depressed in the presence of hSK1YP. Thus, the effect of hSK1YP on rSK3 currents strongly suggests an interaction occurs between these two channel subunits. However, this result does not prove that hSK1 and rSK3 can co-assemble to form functional channels at the cell surface. In an attempt to detect the presence of functional heteromeric channels we therefore used the change-of-function strategy described in the next section.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To further examine SK subunit assembly patterns in vitro, we have utilized both dominant negative and change-of-function approaches. We have been able to demonstrate that in mammalian cells, as in oocytes (15), hSK1 and rSK2 subunits co-assemble. It is interesting that this same pattern of behavior is seen despite dramatic differences in the reported pharmacology of hSK1. Whatever mechanism is responsible for the differences in SK1 observed in these two systems, it does not appear to disrupt SK1/SK2 subunit associations.
An additional interesting feature that emerges from our experiments is that overexpression of the hSK1 dominant negative does not substantially inhibit IK1 (SK4) currents. Although many more experiments are needed, it might eventually be shown that SK and IK channel subunits do not coassemble in any combination. This is potentially an important point when it comes to explaining the diverse range of SK channel pharmacology reported in various tissue preparations (21). If a relatively small number of functional SK channel subunit combinations occur then perhaps SK channels rely on auxiliary 
subunits for diversity (22–24).
Perhaps the most interesting aspect of our data concerns the interaction between rSK1 and rSK3. It appears that rSK1/rSK3 subunit assembly dramatically reduces the number of functional channels at the cell surface although a small current remains (when the cDNAs are transfected in a 2:1 ratio). Similar results have previously been reported in the more extensive studies of Kv potassium channels. Silent channel subunits such as Kv8.1 and Kv9.1 can, presumably by subunit co-assembly, reduce or even abolish current otherwise produced by expression of Kv3.4 (25, 26). In some of this work there is evidence that although the silent subunits cause a reduction in current, channels reaching the cell surface have altered gating kinetics and are therefore likely to be heteromeric assemblies (26, 27). However, when we examined the remaining current seen upon co-transfection of rSK1 with the TEA-sensitive mutant rSK3VF, we found that the sensitivity of the current to block by TEA was the same as for SK3VF channels expressed alone suggesting that there was no formation of functional heteromeric channels at the cell surface. (In keeping with this idea our preliminary experiments suggest that co-expression of rSK1YP, which is not expected to form functional channels, causes a 70 ± 7% inhibition of rSK3 current,2 not significantly different from wildtype rSK1.) Thus, the rSK1/rSK3 interaction may be very similar to that reported for the interaction between the silent Kv9.1 subunit and the fully functional Kv3.4 subunit. In this case, co-expression of the silent Kv9.1 subunit reduces Kv3.4 currents by >80% with no apparent change in the gating properties of channels at the cell surface, implying that they are probably homotetramers of Kv3.4 (26). In contrast to the behavior seen with rSK3, we have recently shown that the opposite behavior occurs when rSK1 is co-expressed with rSK2. In this case rSK1 can interact with rSK2 to augment the levels of functional SK channels (16). It thus seems that the precise combination of subunits is critical in determining whether the complex is functional at the cell surface. This is perhaps most striking for the interaction between hSK1 and rSK3 subunits. The human homologue of SK1, unlike its rat counterpart, produces functional channels when expressed alone. SK3 also produces functional channels and one might expect therefore, that if hSK1 and SK3 co-assemble the resulting channels would, similarly, be functional and at the cell surface. However, co-expression of the TEA-sensitive rSK3VF with hSK1 led to currents with TEA sensitivity not different from that of homomeric hSK1. This finding suggests that rSK3VF subunits made little or no contribution to the observed channels.
As a first step toward understanding the mechanism of interaction between SK1 and SK3 we used an anti-SK3 antibody to study the distribution of SK3 protein. Cells co-transfected with rSK3 and either human or rat SK1 showed clear staining for SK3 protein. However, in the presence of SK1, the distribution of SK3 subunits was altered with much of the protein appearing to be trapped intracellularly. Thus the interaction between SK1 and SK3 disrupts the normal trafficking of SK3 subunits to the cell surface. In this regard the SK family of potassium channels appears to behave similarly to other K+ channel families where subunit co-assembly changes trafficking patterns (see e.g. Ref. 28).
In summary, both SK1/SK2 and SK2/SK3 heteromeric complexes give rise to functional cell surface channels in mammalian cell lines, while IK1/SK1 subunits do not appear to co-assemble. The interaction between SK1 and SK3 suppresses channel activity, possibly because this subunit combination remains trapped intracellularly. The interaction between hSK1 and rSK3 is particularly interesting in this regard because it suggests either that the assembly of this complex might hide an export signal present on both subunits (since both reach the cell surface expressed alone), or that the co-assembly of subunits creates a new, overriding retention signal. These findings thus add to the already complicated set of trafficking interactions known to be involved in distributing potassium channel complexes to the cell surface (29–31) and emphasize the importance of SK subunit combinations in the trafficking process.
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FOOTNOTES |
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Recipient of a MRC/GSK collaborative research studentship.
To whom correspondence should be addressed: Dept. of Pharmacology, University College London, London WC1E 6BT, United Kingdom. E-mail: g.moss{at}ucl.ac.uk.
1 The abbreviations used are: SK, small conductance calcium-activated potassium; PBS, phosphate-buffered saline; ABS, antibody blocking solution; GFP, green fluorescent protein; TEA, tetraethylammonium chloride.
2 G. W. J. Moss, A. S. Monaghan, and Y. A. Shah, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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). The effect of UCL 1848 on homomerically expressed rSK3VK was tested at a single concentration of 300 nM (
). The Hill equation was fitted to the data for rSK2 alone yielding an estimate for the IC50 of 110 ± 26 pM. Each point is the mean of 3–5 observations. Vertical bars indicate S.E.
). The lines are fits of the Hill equation with IC50 values of 14.1 ± 1.0 mM, 0.31 ± 0.07 mM, and 16.0 ± 1.5 mM, respectively.
). The Hill equation fits the data with IC50 values of 8.6 ± 1.6, 0.31 ± 0.07, and 0.24 ± 0.02 mM, respectively. Each data point is the mean of three or more experiments.
) rSK1 with rSK3.