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Originally published In Press as doi:10.1074/jbc.M302091200 on May 6, 2003

J. Biol. Chem., Vol. 278, Issue 28, 25940-25946, July 11, 2003
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Small Conductance Ca2+-activated K+ Channels and Calmodulin

CELL SURFACE EXPRESSION AND GATING*

Wei-Sheng Lee {ddagger}, Thu Jennifer Ngo-Anh {ddagger}, Andrew Bruening-Wright {ddagger}, James Maylie § and John P. Adelman {ddagger} 

From the §Department of Obstetrics and Gynecology and {ddagger}Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239

Received for publication, February 27, 2003 , and in revised form, April 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming {alpha} subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small conductance Ca2+-activated K+ channels (SK channels)1 are fundamental components of cell excitability. SK channels are voltage-independent and activated by elevated intracellular Ca2+ levels that occur during an action potential. In many neurons, SK channels remain open after the action potential and contribute to a post-hyperpolarization, thereby influencing interspike interval and burst duration (1–4). SK channels are also important in peripheral tissues, regulating hormone release from gland cells (5, 6) and smooth muscle tone (7–9).

Four genes comprise the SK channel gene family. SK1, 2, and 3, are expressed in the central nervous system and peripheral tissues, whereas expression of the structurally and functionally similar intermediate conductance channel, IK1, is limited to peripheral tissues (10–12). SK and IK channel {alpha} subunits share the serpentine architecture of voltage-gated K+ channels, each bearing six transmembrane domains, with the N and C termini residing within the cell. The SK subunits are highly homologous but vary in their extreme N- and C-terminal domains. The only striking primary sequence homology between SK channel subunits and other K+ channels is in the pore region located between the fifth and sixth transmembrane domains.

Functional SK channels are heteromeric complexes of four pore-forming {alpha} subunits and calmodulin (CaM) that mediates Ca2+ gating. In inside-out patches containing cloned SK channels, Ca2+-dependent gating persists in the absence of applied CaM, suggesting that CaM is constitutively bound to the native complex (13). Structure-function studies are consistent with this model and have shown that the interaction occurs at the CaM binding domain (CaMBD), a highly conserved stretch of 92 amino acids residing in the proximal region of the intracellular C terminus of the {alpha} subunits (14, 15). The structure of the complex between the CaMBD and Ca2+-CaM partitions CaM into distinct functional domains. Through interactions with the CaMBD, CaM adopts an extended conformation with the globular N- and C-lobes that harbor the E-F hand motifs separated by an elongated linker region. Ca2+ binding to the N-lobe E-F hands 1 and 2 of CaM is necessary and sufficient for Ca2+ gating (14, 16). Residues in the linker domain and the C-lobe maintain Ca2+-independent interactions, including salt bridges between Arg-464 and Lys-467 on the CaMBD and Glu-84 and Glu-87 on CaM. Indeed, the spatial orientation of the residues in E-F hands 3 and 4, usually the higher affinity Ca2+ binding sites, is disrupted by extensive interactions with CaMBD residues and cannot adopt a chelating configuration. Therefore, it is likely that these interactions account for the constitutive association between the proteins (16).

The constitutive association between the channel subunits and CaM permits rapid gating in response to Ca2+ (13, 17). To determine whether the constitutive association between the CaMBD and CaM is required for SK channel gating, mutations that disrupt the constitutive interaction were introduced into SK2. Expression studies show that SK channels can undergo Ca2+-CaM-dependent gating in the absence of a constitutive association with CaM. Surprisingly, Ca2+-independent interactions with CaM are required for cell surface expression of SK channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Biology—Proteins expressed in transfected cells were cloned in the cytomegalovirus-based vector, pJPA. Site-directed mutagenesis was performed using Pfu DNA polymerase (Stratagene, La Jolla, CA). The tandem triple myc epitope (EQKLISEEDL) was inserted at the S3-S4 loop of rSK2 using complementary oligonucleotides cloned into a BamHI site that had been introduced by site-directed mutagenesis at position 246 of rSK2. All sequences were verified by DNA sequence analysis.

Electrophysiology—COSm6 or HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (all from Invitrogen, Carlsbad, CA). Cells were transfected with pJPA expression plasmids encoding CD4, the indicated SK2 channel, and CaM (ratios of DNAs were 1:8:8, respectively) using calcium phosphate for HEK293 cells or lipofection (Qiagen, Valencia, CA) for COSm6 cells. Recordings were performed at room temperature 1–3 days after transfection. Transfected cells were identified by CD4 antibody-coated micorspheres (Dynabeads, M-450 CD4, Dynal, Oslo, Norway). When filled, pipettes prepared from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) had resistances of 1.8–3 M{Omega}. Voltage-clamp recordings were performed with an Axopatch-1B patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 5 kHz (–3 db). For whole-cell recordings pipettes were filled with (in mM) 140 KCl, 10 HEPES, 1 MgCl2, 10 EGTA, with pH adjusted to 7.2 with KOH, after adding CaCl2 to 100 µM. The bath solution was 30 KCl, 110 NaCl, 10 HEPES, 1 MgCl2, 1 CaCl2 (with pH adjusted to 7.2 with NaOH). For excised patch recordings, the pipette solution was (in mM) 30 KOH, 120 NaOH, 10 HEPES, and 1 MgCl2, with pH adjusted to 7.2 with methanesulfonic acid. Excised patches were superfused with an intracellular solution containing (in mM): 150 KOH, 10 HEPES, and 1 EGTA, supplemented with CaCl2, with pH adjusted to 7.2 with methanesulfonic acid; the amount of CaCl2 required to yield the indicated concentrations was calculated according to Fabiato and Fabiato (1979). Current amplitudes were measured at –80 mV unless otherwise indicated.

Rat CaM and the indicated mutants were cloned into pET23b, expressed in BL21 (DE3), and purified on a low substitution phenyl-Sepharose column (Amersham Biosciences, Piscataway, NJ). CaMs were added to the bath solution at 10 µM immediately prior to use. The SK-MLCK M13 peptide (KRRWKKNFIAVSAANRFKKISSSGAL) was synthesized by Genemed Synthesis (South San Francisco, CA).

Immunocytochemistry—COSm6 cells were grown to ~15% confluency in a 60-mm dish on microscope cover glasses and incubated for 5 h with the transfection mixture of 2.75 µg of DNA (ratio of GFP:SK2: CaM, 1:5:5) in 1 ml of DMEM and 8 µl of DMRIE-C reagent (Invitrogen) in another 1 ml of DMEM. The mixture was incubated at room temperature for 20 min prior to cell treatment. After transfection, cells were washed and fed with complete medium and incubated at 37 °C in a 5% CO2. Immunocytochemistry was performed 1–2 days post-transfection.

Non-permeabilized immunostaining was performed by incubating the cells at 37 °C with 1:250 dilution of anti-myc monoclonal antibody (Invitrogen) in complete medium for 1 h. After three washes in complete medium and two washes in PBS+ (1x phosphate-buffered saline containing 1 mM MgCl2 and 0.1 mM CaCl2), cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After quenching with two washes with 50 mM NH4Cl in PBS+, cells were washed once with PBS+. Nonspecific binding was then blocked by incubating the cells with 10% bovine serum albumin (BSA) in PBS+ at room temperature for 30 min. The excess BSA was removed, and the secondary antibody (1:500 dilution of Texas Red-conjugated horse anti-mouse IgG (H+L), Vector, Burlingame, CA) was applied at 4 °C overnight. Cells were then washed three times with PBS+ and mounted (ProLong Antifade Kit, Molecular Probes, Eugene, OR) for imaging.

For permeabilized labeling, cells were washed with PBS+ and fixed with 4% paraformaldehyde at 4 °C for 30 min. After washing three times with ice-cold PBS+, cells were permeabilized with 0.2% Triton X-100 in PBS+ at room temperature for 15 min. To remove excess Triton X-100, cells were washed five times with PBS+ at room temperature. Nonspecific binding was then blocked by incubating the cells with 10% BSA in PBS+ at room temperature for 30 min, and primary antibody was then added and incubated at 4 °C overnight. The next day, the cells were washed and incubated with the secondary antibody at room temperature for 1 h. Cells were washed again, and mounting was performed as described for non-permeabilized cells. Images were acquired with epifluorescence using an optical microscope (Axioplan2, Zeiss, Thornwood, NY) and the program OpenLab (Improvision, Lexington, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SK2:64/67 Channels Require Cotransfected CaM for Function—The crystal structure of the CaMBD·Ca2+-CaM complex from SK2 revealed strong electrostatic contacts between the SK channel CaMBD residues Arg-464 and Lys-467 and CaM Glu-84 and Glu-87 in the CaM linker region close to the C-lobe implicated in constitutive CaM binding (16). Consistent with this observation, the CaMBD peptide harboring the double charge reversal R464E/K467E did not retain CaM in Ca2+-free pull-down assays, whereas the Ca2+-dependent interaction was still detected (14). To further investigate the role of the constitutive interaction between SK channels and CaM, whole-cell recordings were performed from cells transiently transfected with SK2 wild type or SK2:64/67 mutant channels, with or without cotransfected CaM (Fig. 1). Five minutes after whole-cell patch formation with Ca2+ (100 µM) in the patch pipette, cells transfected with wild type SK2 and recorded in asymmetrical K+ showed large currents (–4.4 ± 0.5 nA, n = 5, Fig. 1A) that were reversed in response to voltage ramp commands close to the predicted K+ reversal potential (–40 mV). Cotransfection with CaM did not obviously affect current responses (–4.4 ± 1.0 nA, n = 5, Fig. 1B). Point mutation charge reversals at either of Arg-464 or Lys-467 resulted in functional channels that were not obviously different from wild type (not shown), whereas only small currents that were not different from mock transfected cells were recorded from cells transfected with SK2:64/67 (–30.0 ± 10.0 pA, n = 5, Fig. 1C). However, when SK2:64/67 was cotransfected with CaM, robust currents were recorded within 1 min of whole-cell patch formation and increased to a steady state by 5 min (–5.3 ± 1.0 nA, n = 5, Fig. 1D), indicating that SK2:64/67 channels retain an ability to interact with Ca2+-CaM.



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  		FIG. 1.
SK2:64/67 requires coexpressed CaM for function. A–D, representative whole-cell voltage clamp recordings from HEK293 cells transfected with the indicated expression plasmids, 5 min after whole-cell patch formation. To activate SK channels, Ca2+ (100 µM) was dialyzed into the cell through the patch pipette. The traces show responses to 2-s voltage ramp commands from –80 to 80 mV. E, whole-cell current amplitudes ± S.E. measured at –80 mV (n = 5).

 

Exogenous CaM Rescues SK2:64/67 Activity in Excised Patches—To more closely examine the interaction between CaM and SK2:64/67, inside-out patches were excised from transiently transfected COS cells into Ca2+-containing solution (10 µM, EK = –40 mV). In the absence of cotransfected CaM, currents measured at –80 mV (–31.3 ± 5.7 pA, n = 13) were not different from mock transfected cells (–23.3 ± 6.8 pA, n = 4, p = 0.5, unpaired t test). In contrast, patches from cells cotransfected with SK2:64/67 and CaM yielded channel activity immediately after excision (–638.3 ± 109.3 pA, n = 19), but channel activity diminished even while maintaining the patches in Ca2+ solution such that within 3 min the current measured at –80 mV decayed to 40.2 ± 5.7% of the initial current amplitudes (–294.9 ± 81.8 pA, n = 19). If patches were excised into Ca2+ solution (–887.5 ± 214.7 pA, n = 10) and then exposed to Ca2+-free solution (0 Ca2+) for 1 min, SK currents disappeared (–38.4 ± 12.3 pA, n = 10), and, different from wild type, a return to Ca2+ solution did not re-evoke SK currents (–42.0 ± 9.9 pA, n = 10).

One possible reason for the rapid decay of SK2:64/67 channel activity in patches and the inability to repeatedly activate the channels by exposure to Ca2+ is that the channels are weakly associated with CaM prior to and immediately after excision into Ca2+ solution, but channel activity is lost as CaM dissociates from the channels. To test this possibility, patches from cotransfected cells were excised into Ca2+ solution, and currents were evoked as described above. Ca2+-dependent channel activity was abolished by exposure to Ca2+-free solution. However, when exposed to Ca2+ solution additionally containing 10 µM CaM (Ca2+-CaM solution), 56.7 ± 12.8% of the initial channel activity was reconstituted (n = 10, Fig. 2, A and B). Upon re-exposure to 0 Ca2+ solution and return to Ca2+ solution, currents could not be evoked, nor were currents evoked by exposure to CaM solution in the absence of Ca2+.



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  		FIG. 2.
Ca2+-CaM rescues SK2:64/67 in excised patches. A, currents recorded from a representative inside-out patch excised from a COS cell cotransfected with SK2:64/67 and CaM. The patch was excised into Ca2+ solution (10 µM, trace 1), then exposed to 0 Ca2+ solution (trace 2), and returned to Ca2+ solution (trace 3), before Ca2+-CaM (10 µM) was applied (trace 4), rescuing channel activity. B, diary plot of the current measured at –80 mV from the patch shown in A. The numbers correspond to the current responses shown on the left. C, diary plot of the currents measured at –80 mV from a separate patch exposed to three sequential applications of Ca2+-CaM (10 µM) separated by exposure to Ca2+ solution (10 µM) lacking CaM.

 

The time course of Ca2+-CaM binding and unbinding was examined by three sequential applications of Ca2+-CaM solution separated by exposure to Ca2+ solution lacking CaM (Fig. 2C). The amplitude of the currents sampled at –80 mV increased upon addition of Ca2+-CaM and decreased upon switching to Ca2+ solution without CaM. Fitting the data with single exponentials yielded time constants of 1.2 ± 0.2, 1.7 ± 0.3, and 1.8 ± 0.4 min for Ca2+-CaM association and 0.7 ± 0.1, 1.4 ± 0.4, and 2.1 ± 0.8 min for Ca2+-CaM dissociation (n = 7). The maximum current amplitudes decreased over the course of the experiment, consistent with channel rundown (18).

To determine whether CaM could associate with SK2:64/67 channels in the absence of Ca2+, patches were excised into Ca2+ solution, verifying functional channels, and then exposed to 0 Ca2+ solution during which time CaM dissociated as confirmed by subsequent exposure to Ca2+ solution. Patches were then exposed to Ca2+-free CaM (10 µM) for 5 min. Upon switching to Ca2+ solution lacking CaM, currents were rapidly evoked, indicating that CaM had assembled with the channels during the Ca2+-free incubation (Fig. 3A). After CaM dissociation and decay of the currents, reapplication of Ca2+-CaM for 5 min again rescued the currents (Fig. 3B). The percentage of the current evoked upon exposure to Ca2+ following Ca2+-free CaM compared with the current subsequently evoked by Ca2+ CaM was 29.7 ± 3.8% (n = 4). This result shows that Ca2+-free CaM can associate with SK2:64/67 channels and suggests that the CaM affinity is increased by the presence of Ca2+.



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  		FIG. 3.
Ca2+-free CaM associates with SK2:64/67. Top, continuous recording at –80 mV from an inside-out patch containing SK2:64/67 channels. After a 5-min application of CaM in the absence of Ca2+, currents were evoked upon exposure to a solution lacking CaM but containing 10 µM Ca2+ (corresponds to point 5, below). Bottom, diary plot of the patch shown above. The patch was excised into Ca2+ solution (10 µM, 1), then exposed to 0 Ca2+ solution (2), and returned to Ca2+ solution (3), before Ca2+-free CaM (10 µM) was applied (4). Exposure to Ca2+ without CaM (5) evoked a current that decayed within 1.5 min. Channel activity was rescued (6) by a second application of Ca2+-CaM.

 

CaM antagonists such as the M13 peptide (19) or calmidazolium (20) do not interfere with the Ca2+-dependent gating process of wild type SK2 (13). It is likely that the constitutive interaction of CaM with SK2 does not present an exposed hydrophobic domain on CaM for antagonist binding (16). However, the interaction between SK2:64/67 and CaM is less stable in the absence of Ca2+. Therefore, patches containing wild type SK2 or SK2:64/67 channels were exposed to Ca2+-CaM solution (1.2 ± 0.3 nA for wild type, n = 9; –486.0 ± 187.2 pA for SK2:64/67, n = 10) and then exposed to the same solution additionally containing the M13 peptide (100 µM). In contrast to wild type SK2 channels, the M13 peptide blocked the ability of Ca2+-CaM to activate SK2:64/67 channels (–1.1 ± 0.3 nA for wild type, n = 9; –32.4 ± 7.9 pA for SK2:64/67; n = 10). Interestingly, the rate of current inhibition (0.28 ± 0.04 min, n = 8) was greater than removal of CaM from the bath solution (see Fig. 2C) suggesting that the excess M13 peptide enhanced the dissociation of SK2:64/67-bound CaM.

Compensatory Mutations in CaM Restore Association with SK2:64/67 Channels—To test whether the double charge reversal E84R/E87K in CaM (CaM:84/87) might compensate for the R464E/K467E double charge reversal in SK2, cells were cotransfected with SK2:64/67 and CaM:84/87. Inside-out patches exposed to Ca2+, displayed SK currents (–1.7 ± 0.6 nA, n = 8) that disappeared upon subsequent exposure to 0 Ca2+ solution (–43.3 ± 12.2 pA, n = 8). Distinctly different from cotransfection of SK2:64/67 with wild type CaM, returning the patch to Ca2+ solution reactivated the channels, rescuing 62.2 ± 12.0% of the initial current (Fig. 4).



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  		FIG. 4.
CaM:84/87 compensates for SK2:64/67. In A: Left, currents recorded from a representative inside-out patch excised from a COS cell cotransfected with SK2:64/67 and CaM:84/87. The patch was excised into Ca2+ solution (10 µM, trace 1), then exposed to 0 Ca2+ solution (trace 2), and returned to Ca2+ solution (trace 3). Ca2+ alone is sufficient to reactivate the channels; application of Ca2+-CaM is not required. Right, diary plot of the current measured at –80 mV from the patch shown on the left. The numbers correspond to the current responses shown on the left. In B: left, currents recorded from a representative inside-out patch excised from a COS cell cotransfected with wild type SK2 and CaM:84/87. The patch was excised into Ca2+ solution (10 µM, trace 1), then exposed to 0 Ca2+ solution, returned to Ca2+ solution (trace 2), and exposed to Ca2+-CaM (trace 3) before returning to 0 Ca2+ solution and exposure to Ca2+ (trace 5). Right, diary plot of the current measured at –80 mV from the patch shown on the left. The numbers correspond to the current responses shown on the left.

 

Cotransfection of wild type SK2 with CaM:84/87 resulted in channels that activated upon patch excision into Ca2+ solution (–594.2 ± 225.4 pA, n = 8) and rapidly decreased without CaM in the bath solution. Unlike coexpression of SK2:64/67 with wild type CaM, currents did not completely disappear, plateauing at 53.2 ± 6.4% of the initial current. This current may reflect channels that have assembled with wild type CaM. Following exposure to 0 Ca2+ solution, re-exposure to Ca2+ solution evoked 62.2 ± 5.3% of the initial current. Subsequent exposure to Ca2+-CaM solution rescued the currents (–740.7 ± 317.6, n = 8) (Fig. 4B). These results suggest that the E84R/E87K charge reversals in CaM destabilize the interaction between SK2 and CaM, and that in patches, CaM:84/87 is lost and may be replaced by wild type CaM.

Ca2+ Dependence—Coexpression studies with wild type SK2 and CaMs harboring point mutations in the E-F hand domains showed that E-F hands 1 and 2 are necessary and sufficient for Ca2+ gating. Mutations in E-F hands 3 and 4 (CaM3,4) that abolish Ca2+ binding do not alter Ca2+ sensitivity, whereas mutations in either E-F hands 1 or 2 (CaM1 or CaM2) shift the sensitivity from ~0.5 to ~1 µM, and the double mutant, CaM1,2, eliminated Ca2+ gating. Functional studies were confirmed by the crystal structure of the CaMBD·Ca2+-CaM complex that showed E-F hands 1 and 2 occupied by Ca2+ ions, whereas E-F hands 3 and 4 were uncalcified (14, 16). To examine the Ca2+ dependence of SK2:64/67 channels, and whether exogenous application of Ca2+-CaM reconstituted SK channel gating similar to wild type, cells were cotransfected with either WT CaM, CaM1,2, CaM3,4, CaM1,2,3,4, or CaM:84/87. 1–3 days later, patches were excised into an internal solution containing the same purified recombinant CaM (10 µM), and Ca2+ dose responses were performed by changing between internal solutions containing CaM with varying concentrations of Ca2+ (Fig. 5). For either wild type or SK2:64/67, neither CaM1,2 nor CaM1,2,3,4 supported Ca2+ gating (not shown). The Ca2+ sensitivity of SK2:64/67 with wild type CaM was right-shifted (Kd = 0.82 ± 0.07 µM, n = 5) compared with wild type channels (Kd = 0.51 ± 0.02 µM, n = 7, p = 0.001, unpaired t test). Application of CaM:84/87 to wild type SK2 channels right-shifted the Ca2+ dose response (Kd = 0.82 ± 0.06 µM, n = 9) to the same extent as SK2:64/67 was right-shifted in the presence of wild type CaM (p = 0.97, unpaired t test). Coexpression of SK2:64/67 with CaM:84/87 rescued stable currents patches (see above) but with reduced Ca2+ affinity (Kd = 1.38 ± 0.06 µM, n = 8). Application of CaM3,4 to either wild type or SK2:64/67 slightly left-shifted the apparent affinity compared with wild type CaM (not shown).



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  		FIG. 5.
Ca2+ dose-responses. Top, normalized Ca2+ dose-response relationships. The indicated SK2 channel and CaM were coexpressed, and the same purified recombinant CaM was applied with different Ca2+ solutions to the inside face of patches. Relative current amplitudes measured at –80 mV from n ≥ 5 patches for each combination of channel and CaM were averaged and plotted versus the intracellular Ca2+ concentration. The averaged data were fitted with a Hill equation (continuous lines) yielding an EC50 (µM) and (Hill coefficient) of 0.51 ± 0.02 (5.6) for wild type SK2 with wild type CaM, 0.82 ± 0.07 (7.1) for SK2:64/67 with wild type CaM, 0.82 ± 0.06 (4.2) for wild type SK2 with CaM:84/87, and 1.38 ± 0.07 (6.4) for SK2:64/67 with CaM:84/87. Bottom, currents measured in response to voltage ramps in representative patches from cells coexpressing the indicated SK2 channel and CaM for 0, 0.6, or 1, and 10 µM Ca2+.

 

CaM Is Required for SK Channel Cell Surface Localization— Application of Ca2+ or Ca2+-CaM to patches from cells transfected with SK2:64/67 alone did not yield currents, whereas patches from cells cotransfected with CaM yielded robust channel activity. These results suggest that, in the absence of cotransfected CaM, SK2:64/67 channels may not be present on the cell surface. To test this possibility, three tandem copies of the myc epitope were inserted into the extracellular loop between transmembrane domains 3 and 4 in SK2 and SK2:64/67. Cells were transfected with the channel alone or in combination with CaM and then examined by immunocytochemistry with a monoclonal anti-myc antibody, either with or without permeabilizing the cells; cotransfected GFP was used to identify transfected cells. For SK2 with or without cotransfected CaM, channel protein was detected on the cell surface when cells were not permeabilized as well as in intracellular organelles after permeabilization (not shown). In contrast, SK2: 64/67 protein was detected on the cell surface only when cotransfected with CaM, even though the protein was highly expressed as evidenced by the strong intracellular organelle staining in permeabilized cells (Fig. 6, top, middle panels).



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  		FIG. 6.
CaM is required for surface expression. Immunocytochemistry of COS cells transfected with the indicated combinations of SK2:64/67, CaM, and a GFP expression plasmid. SK2:64/67 harbors three tandem copies of the myc epitope in the external loop between transmembrane domains 3 and 4. For each panel, transfected cells were visualized by expression of GFP (G; left), channel protein was detected with an anti-myc mouse monoclonal antibody and visualized by Texas Red-conjugated horse anti-mouse secondary antibody (R; middle), and the signals were merged (M; right). In each case, cells were examined either without (–) or with (+) the membrane permeabilization. Top, SK2:64/67 was not detected on the cell surface (–), but channel protein was detected inside permeabilized cells (+). SK2:64/67 was detected on the cell surface (–) as well as inside the cell (+) when transfected with wild type CaM (middle) or the Ca2+-independent CaM mutant, CaM1,2,3,4 (bottom).

 

To determine whether requires Ca2+-CaM, cells were cotransfected with wild type SK2 or SK2:64/67 and CaM1,2,3,4, a Ca2+-independent form of CaM (13). When cotransfected with CaM1,2,3,4, SK2:64/67 subunits were detected in the plasma membrane, demonstrating that Ca2+ binding to CaM is not essential for cell surface expression (Fig. 6, lower panels). Patches from these cells were excised into Ca2+ solution but did not yield SK currents (–31.4 ± 4.5 pA, n = 9). However, application of exogenous wild type Ca2+-CaM resulted in channel activation (–488.8 ± 85.1 pA, n = 9) suggesting that the channels were in the plasma membrane and initially associated with non-functional CaM1,2,3,4, which was replaced by the exogenous functional Ca2+-CaM. Consistent with this idea, patches from cells cotransfected with wild type SK2 and CaM1,2,3,4 were detected in the plasma membrane, and excision into Ca2+ solution did not result in channel activation (–13.3 ± 4.4 pA, n = 4), nor could channel activity be restored by exogenous Ca2+-CaM (–11.1 ± 3.0 pA, n = 4). Therefore, wild type SK2 channels retain constitutively bound CaM1,2,3,4, which cannot bind Ca2+ and cannot be replaced by wild type CaM but are properly trafficked to the cell surface.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here show that Ca2+-independent interactions between the CaMBD and CaM are essential for cell surface expression and that the constitutive binding between the pore-forming {alpha} subunits of SK channels and CaM is not required for channel gating. The crystal structure of the complex between the CaMBD and Ca2+-CaM showed strong interactions between Arg-464 and Lys-467 on the channel and Glu-84 and Glu-87 on CaM, in the region implicated in constitutive association (16). This was supported by the lack of channel function when the double mutant was expressed. In addition, the purified CaMBD R464E/K467E peptide failed to retain purified CaM in pull-down assays in the absence of Ca2+ (14). These results show that the CaMBD R464E/K467E is different from wild type in its ability to retain CaM, but they do not distinguish between a complete lack of binding or a weakened affinity.

The crystal structure of the CaMBD·Ca2+-CaM and functional studies with mutant CaMs (14, 16) suggested a separation of function between the two lobes of CaM with the C-lobe mediating many of the Ca2+-independent interactions with CaMBD. Indeed, the shape of E-F hands 3 and 4 are altered through the multiple interactions with CaMBD residues and no longer coordinate Ca2+ ions. The N-lobe E-F hands 1 and 2 bound Ca2+ and were comparable to other Ca2+-CaM substrate structures (21–23). Therefore, if the double mutation R464E/K467E eliminated all Ca2+-independent interactions, Ca2+ gating in this channel might be mediated solely by interactions with either Ca2+-loaded N- or C-lobes. However, application of mutant CaMs with different combinations of intact E-F hands to SK2:64/67 channels showed that, just as for wild type channels, E-F hands 1 and 2 are required, whereas E-F hands 3 and 4 are dispensable. This implies that the double mutation R464E/K467 weakens the Ca2+-independent interactions between the channel subunit and CaM and that the reconstituted channels interact with exogenously applied CaM similar to that of wild type but with a reduced Ca2+ sensitivity. Therefore, it is likely that overexpressed CaM rescues whole cell SK2:64/67 channels and surface expression by overcoming the weakened affinity.

The compensatory mutations in CaM reconstituted tighter binding between the two double mutants, SK2:64/67 and CaM: 84/87, presumably by reinstating salt bridges between these positions. However, the reconstituted channels have reduced Ca2+ sensitivity, with apparent Kd values even more right-shifted than SK2:64/67 with WT CaM suggesting that, although the complex is stabilized, the conformational changes that open the channel gate subsequent to Ca2+ binding are compromised.

A role for CaM in SK channel trafficking was also found. Immunocytochemistry clearly demonstrated surface expression of SK2:64/67 only with cotransfected CaM, and functional studies showed that the channels carried associated CaM. Joiner et al. (24) had observed that overexpressing a part of the C-terminal domain of IK1 that included the CaMBD redistributed the channels to the intracellular compartments and overexpressing CaM redeposited them in the plasma membrane. The present results extend the implications for trafficking by showing that the Ca2+-independent interactions between the channel subunits and CaM are sufficient for cell surface expression. Patches from cells cotransfected with SK2:64/67 channels and CaM1,2,3,4 did not show channel activity when excised into Ca2+ solution, but channel activity was reconstituted upon subsequent application of Ca2+-CaM. Immunocytochemistry verified surface expression of SK2:64/67 channels when cotransfected with CaM1,2,3,4. This result is consistent with the ability of Ca2+-free CaM to associate with the channels in excised patches.

Because the CaM-dependent gating mechanism was described for SK channels, a variety of other channels have been shown to undergo Ca2+-free and Ca2+-dependent CaM interactions that alter their functions. For example, Ca2+-free CaM binds to an I-Q motif in the intracellular C-terminal domains of L-, N-, and P/Q-type voltage-gated Ca2+ channels (25–28), and similar to SK channels the different lobes of CaM mediate different functions (29). CaM binds to and modulates specific isoforms of voltage-gated Na+ channels (30, 31), cyclic nucleotide-gated channels (32, 33), and CaM is an auxiliary subunit of human gene transient receptor potential channels (34, 35). CaM also binds to and regulates the function of several ionotropic receptors, Ca2+ release channels, and TRP channels (see 36–38). CaM is not the only E-F hand Ca2+-binding protein that directly regulates ion channel function. Members of the Kv4 family of voltage-gated K+ channels interact with K-channel-interacting proteins that endow important biophysical properties as well as regulating trafficking to the plasma membrane (39). For SK channels, it remains to be determined just how the binding of CaM influences trafficking. However, the SK channels contain a conserved RKR motif in the intracellular N terminus, immediately preceding the first transmembrane domain. An analogous situation exists for functional KATP channels in which the channel forming Kir 6.2 subunits require association with SUR1 for surface expression. In this case, trafficking is regulated by the RKR endoplasmic reticulum retention signals present in each of the partner subunits that is exposed prior to co-assembly and buried once the two subunits form the macromolecular complex (40). However, mutagenesis of the RKR motif to AAR in SK2:64/67 did not result in surface expression in the absence of cotransfected CaM, although channel subunits were detected inside the cell (not shown).

CaM is highly expressed in almost all cell types, yet the concentrations and subcellular localization of free CaM may vary dramatically depending upon the state of phosphorylation, anchoring to the plasma membrane, or association with CaM storage proteins such as GAP-43 (41–43). Moreover, distinct CaM pools may be differentially mobilized, transiting long cellular distances, from dendrites to the nucleus (44). In addition, local CaM concentrations may also be translationally regulated as CaM mRNA is differentially distributed, a process that likely reflects the conservation of three non-allelic mammalian CaM genes encoding identical proteins but distinct 5' and 3' non-coding sequences. For example, one pool of CaM mRNA, derived from a specific CaM gene (CALM1) is abundant in the apical dendrites of cerebellar pyramidal cells and may give rise to local reservoirs of CaM; however, mRNAs derived from CALM1 and CALM2 genes are found in neurite outgrowths in nerve growth factor-stimulated PC12 cells, and CALM3-derived transcripts reside within the cell body (45–47). Base upon these factors, and the number and concentrations of CaM-binding proteins, it is possible that CaM availability is rate-limiting for SK channel surface expression (43). In this case, metabolic processes that alter the concentrations of free CaM may dynamically regulate SK current density and cell excitability.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-5450; Fax: 503-494-4353; E-mail: adelman{at}ohsu.edu.

1 The abbreviations used are: SK channel, small conductance Ca2+-activated potassium channel; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GFP, green fluorescent protein; CaM, calmodulin; IK channel, intermediate conductance Ca2+-activated potassium channel; CaMBD, calmodulin binding domain; SK2:64/67, SK2 R464E/K467E; CaM:84/87, CaM E84R/E87K; CaM1,2, CaM3,4, and CaM1,2,3,4, calmodulins with mutations in the first position of the indicated E-F hands, effectively abolishing Ca2+ binding. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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