From the
Department of Obstetrics and Gynecology and
Vollum Institute, Oregon Health & Science
University, Portland, Oregon 97239
Received for publication, February 27, 2003 , and in revised form, April 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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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
(1012).
SK and IK channel 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
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
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.
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MATERIALS AND METHODS |
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ElectrophysiologyCOSm6 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 13 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
1.83 M. 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).
ImmunocytochemistryCOSm6 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 12 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).
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RESULTS |
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Exogenous CaM Rescues SK2:64/67 Activity in Excised PatchesTo 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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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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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 ChannelsTo 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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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+ DependenceCoexpression 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. 13 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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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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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.
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DISCUSSION |
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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 (2123). 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 (2528), 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 3638). 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 (4143). 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 (4547). 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.
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FOOTNOTES |
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¶ 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.
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REFERENCES |
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