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Published as doi: 10.1096/fj.07-9712com.
 (The FASEB Journal. 2008;22:1135-1143.)
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(The FASEB Journal. 2008;22:1135-1143.)
© 2008 FASEB

Calmodulin regulates the trafficking of KCNQ2 potassium channels

Ainhoa Etxeberria*,1, Paloma Aivar*,1, Jose Angel Rodriguez-Alfaro*,1, Alessandro Alaimo*, Patricia Villacé*, Juan Camilo Gómez-Posada*, Pilar Areso{dagger} and Alvaro Villarroel*,2

* Unidad de Biofísica, CSIC-UPV/EHU,

{dagger} Departamento Farmacología, UPV/EHU, Universidad del País Vasco, Leioa, Spain

2Correspondence: Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain. E-mail: gbxvimua{at}lg.ehu.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca2+-binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild-type channels in the ER. Thus, a direct interaction with Ca2+-calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels’ exit from the ER.—Etxeberria, A., Aivar, P., Rodriguez-Alfaro, J. A., Alaimo, A., Villacé, P., Gómez-Posada, J. C., Areso, P., Villarroel, A. Calmodulin regulates the trafficking of KCNQ2 potassium channels.


Key Words: channelopathies • epilepsy • BFNC • Kv7 • benign familial neonatal convulsions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NEURONAL KCNQ2 AND KCNQ3 potassium channel subunits can constitute the tetrameric voltage-dependent K+ M-channel (1) , a channel that operates in the subthreshold voltage range of action potential generation. Because the activation and deactivation kinetics of the M-channel are slow and the channel does not inactivate, the M-current serves to counterbalance the generation of action potentials. To date, 5 KCNQ subunits have been identified, and, in addition to KCNQ2 and KCNQ3, KCNQ4 and KCNQ5 subunits also can contribute to the formation of M-channels (2) .

The flow of K+ ions through M-channels is fundamental in controlling neuronal excitability, postnatal brain development, and cognitive performance (2 3 4) . The KCNQ2/KCNQ3 channels are preferentially located at the surface of the distal regions and the initial segment of the axons. This distribution reflects the role they fulfill in regulating the propagation of action potentials along the axon (5 6 7 8) and the release of neurotransmitter from the nerve terminal (9) . Mutations in the human KCNQ2 or KCNQ3 genes can provoke benign familial neonatal convulsions (BFNC), a dominantly inherited idiopathic epilepsy (2) . However, apart from 3 mutations in KCNQ3, all the mutations associated with BFNC have been identified in KCNQ2 subunits (2) . Interestingly, mutations that cause a mere 25% reduction in current provoke BFNC (10) , indicating that the attainment of a threshold of M-channel function is essential to maintain normal physiological activity.

The ubiquitous calcium sensor calmodulin (CaM) has been shown to associate with the intracellular C-terminal region of every member of the KCNQ (Kv7) family of K+ channels (11 , 12) . This association mediates the calcium-dependent inhibition of the M-like current produced by KCNQ2/KCNQ3 subunits in heterologous systems (13) . CaM is a small protein present in all eukaryote cells at concentrations ranging from 1 to 10 µM. However, much of the CaM present in resting cells is sequestered, and the free available CaM represents only a small fraction of the total population (varying from 1/20 to 1/1,000) (14) . Structurally, CaM is made up of 2 similar globular domains, the N- and C-lobe, which are connected by a flexible central linker. Each lobe is comprised of 2 helix-loop-helix motifs that constitute the Ca2+-binding EF-hand motifs. The binding of Ca2+ induces a conformational change that results in the modification of the target protein, conferring calcium sensitivity to the activity of many enzymes, pumps, and ion channels (15 , 16) . For instance, CaM is involved in the Ca2+-dependent inactivation of L-type Ca2+ channels and NMDA receptors, and it is essential for the Ca2+-dependent activation of both SK and IK potassium channels. Apart from this Ca2+ sensor function, CaM also has been implicated in the assembly of SK potassium channel subunits (16 17 18) .

CaM currently is believed to be an essential auxiliary subunit of KCNQ2/KCNQ3 channels because KCNQ2 mutants deficient in CaM binding are unable to generate currents when coexpressed with KCNQ3 subunits. However, these mutants retain the ability to assemble with KCNQ3 and do not display obvious defects in targeting to the plasma membrane. Moreover, sequestering CaM reduces the M-current density (12 , 19) .

Here, we tested the hypothesis that CaM may be required for the trafficking of KCNQ2 subunits to the plasma membrane. We found that CaM regulates the exit of KCNQ2 channels from the endoplasmic reticulum (ER) by a mechanism that is independent of subunit heteromerization. These findings represent a novel Ca2+-dependent mechanism to directly regulate the trafficking of a membrane protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular biology
The human KCNQ2 (Y15065) and KCNQ3 (NM004519) cDNAs were kindly provided by Thomas J. Jentsch (ZMNH, Hamburg, Germany) and all deletions, point mutations, and epitope insertions in the KCNQ subunits were constructed by PCR-based mutagenesis. The cDNA encoding rat CaM and the Ca2+-independent form of CaM (CaM1234), in which the first aspartate of each EF hand is replaced by alanine (20) , were provided by the group of John P. Adelman (Vollum Institute, Oregon Health Sciences University, Portland, OR, USA). For immunoprecipitation experiments, we used the KCNQ2 subunit tagged at the N-terminal with a tandem repeat of 5 myc epitopes (MEQKLISEEDLN), the KCNQ3 subunit with a tandem repeat of 2 HA epitopes (YPYDVPDYA), and the CD helices (L531-G656) with a TAP epitope at the C-terminal end (CD-TAP).(21) All constructs were subcloned into pCAGGS, provided by Juan C. de la Torre (the Scripps Research Institute, San Diego, CA, USA). For imaging, KCNQ2 was tagged at the N terminus with mCFP in pSRC5, a low copy number vector, and CaM and Neurogranin were tagged at the N-terminal with YFP and cloned into pCDNA3.1 (Invitrogen, Carlsbad, CA, USA). The ER marker pDsRed2-ER was obtained from Clontech Laboratories Inc. (Palo Alto, CA, USA). Helices A and B from KCNQ2 (A309 to L549) were subcloned into pCDNA3.1 (Invitrogen) and tagged at the N terminus with a tandem repeat of 5 myc epitopes. The Tac-CFP construct was generated using the Tac receptor provided by Dr. Steve Standley (Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, MD, USA), which was cloned into a modified version of the expression vector pEGFP (Clontech), where eGFP has been exchanged for mCFP. The electrophysiological properties recorded in Xenopus oocytes of tagged KCNQ2 and KCNQ3 constructs were indistinguishable from the nontagged equivalents.

Antibodies
The antibodies used here included: a mouse polyclonal antimyc (9E10), a gift of Dr. Sebastian Pons (Instituto de Investigaciones Biomédicas de Barcelona, Spain); a rat monoclonal anti-HA (3F10; Roche Diagnostics, Barcelona, Spain); a mouse monoclonal anti-CaM (Upstate Biotechnology, Lake Placid, NY, USA); a rabbit anti-CBP (Upstate Biotechnology); a mouse monoclonal anti-GFP (clones 7.1 and 13.1; Roche); and a mouse monoclonal anti-{alpha} tubulin (DM1A; Sigma-Aldrich Corp., St Louis, MO, USA). The peroxidase-coupled secondary antibodies used were an anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA, USA), anti-rat IgG (Jackson ImmunoResearch Laboratories Inc., Suffolk, UK), or anti-rabbit IgG (Jackson ImmunoResearch Laboratories).

Electrophysiological measurements
Standard 2-electrode voltage clamp procedures were employed to record currents from Xenopus oocytes (22) . To monitor the assembly of KCNQ2 point mutants with KCNQ3, sensitivity to blockage by TEA was measured (22) . Currents were elicited by an 800 ms depolarizing step from –120 to +50 mV every 20 s. Data were normalized in Excel (Microsoft Corp., Madrid, Spain) and plotted in Sigmaplot (SPSS Corp., Madrid, Spain).

Surface expression
A KCNQ3 subunit tagged with an HA epitope in the extracellular loop connecting the S1 and S2 transmembrane domains was used in chemiluminescent assays of single oocytes (22) .

Cell culture and transfection
HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37°C in 5% CO2. Cells were transiently transfected with cDNAs using calcium phosphate, and the behavior of the constructs expressed was analyzed 24 h after transfection.

Immunoprecipitation
HEK293T cells were solubilized for 30 min at 4°C 24 h after transfection in IP buffer containing Tris-HCl 50 mM, NaCl 150 mM, 1% Triton X-100, EDTA 2 mM, EGTA 5 mM, and protease inhibitors (1x Complete; Roche). The nuclei were pelleted at 500 g for 3 min, followed by centrifugation at 11,000 g for 20 min to remove the insoluble material. Lysates were precleared with 40 µl of equilibrated Protein A sepharose beads (Sigma P3391) for 1 h at 4°C. Anti-myc antibodies were immobilized with 40 µl of equilibrated Protein A beads overnight at 4°C and washed twice with IP buffer. Precleared lysates were incubated with Protein A-anti-myc for 4 h at 4°C and, after 4 washes with IP buffer, immunoprecipitated proteins were recovered after heating at 90°C for 5 min in sodium dodecyl sulfate (SDS) sample buffer.

To immunoprecipitate the CD-TAP protein, HEK293T cells were solubilized in 50 mM Tris-HCl pH 7.5, NaCl 100 mM, EDTA 5 mM, 0.5% Nonidet P-40, and protease inhibitors (1x Complete; Roche). The nuclei and insoluble material was removed as described above. The cell lysates then were incubated with IgG-sepharose (Amersham Biosciences Corp., Barcelona, Spain) for 4 h at 4°C, followed by 10 washes with IPP150 buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 0.1% Nonidet P-40).

Image acquisition and analysis
HEK293T cells were washed with PBS 24 h after transfection using calcium phosphate and then fixed in freshly diluted 3.5% paraformaldehyde (Sigma) for 20 min. Colocalization experiments were performed using a Nikon TE2000-U microscope (Nikon, Melville, NY, USA) equipped for epifluorescence and with a Nikon D-eclipse C1 Si confocal spectral detector, using an x60, 1.45 numerical aperture, oil immersion objective. The fluorescent proteins were excited using a 437 nm laser line, and 32 images were acquired simultaneously at different emission wavelengths between 450 and 610 nm at 5 nm intervals. For each cell, 10 optical sections (approximately 0.6 µm thick) were acquired using a 30 µm diameter pinhole, and cells with similar levels of expression (as judged by brightness) were selected for analysis. The signal from each fluorophore was isolated by performing spectral unmixing with the EZ C1 3.00 software (Nikon). The reference spectra were acquired from cells expressing mCFP-KCNQ2, pDsRed2-ER, or YFP-tagged constructs alone. Each image was analyzed for colocalization using ImageJ software (Version WCIF, National Institutes of Health, USA), using the plug-in to calculate the Manders coefficient (23) . Images were converted to 8-bit, and the perimeter of the cell was drawn manually for analysis. Prior to Manders analysis, the background was automatically subtracted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
More than half of the mutations in KCNQ2 that cause BFNC have been localized to the intracellular C-terminus of this channel subunit. This C-terminal region harbors 4 regions with a high probability of adopting an alpha-helix configuration (helices A–D, Fig. 1 A) and, together, helices A and B constitute a binding site for the ubiquitous cytosolic Ca2+ binding protein CaM. Disruption of this binding site by deleting helix A ({Delta}L339–T358) or B ({Delta}S511–S523) (11) impairs CaM binding, and this interaction is essential for KCNQ channel function (11 , 12) . The introduction of key point mutations into helix A of KCNQ2 (I340E, A343D) abolished apoCaM-binding (Fig. 1B ), and the R353G mutation responsible for the inherited epilepsy BFNC (24) produced an ~40% decrease in the amount of CaM that coimmunoprecipitated with KCNQ2. Because the M current is mainly formed by KCNQ2/KCNQ3 heteromeric channels, the binding of CaM to heteromeric channels containing the KCNQ2 mutant subunits and WT-KCNQ3 was examined (Fig. 1B ). There was an ~60% reduction in the CaM that coimmunoprecipitated with the I340E and A343D heteromeric mutant channels when compared to that obtained with WT-KCNQ2 subunits, whereas the BFNC R353G mutant reduced the signal by ~20%. Since the protein complex was recovered using antibodies to the tagged KCNQ2 subunit, these results indicate that CaM was associated with KCNQ3 in the mutant heterotetramers or, alternatively, to a binding site formed by helix A of KCNQ3 and helix B of KCNQ2.


Figure 1
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  		Figure 1. CaM binding is impaired in KCNQ2 C-terminal point mutants. A) Schematic topological representation of the KCNQ2 channel subunit in which the CaM binding site (11 , 12) and the subunit interacting domain (SID) (27) are indicated. The consensus IQ residues and the 1–5-10 CaM binding motives are shown in bold. The amino acid residues in circles correspond to the residues mutated in this work, and the square indicates the mutation causing BFNC (24) . B) Top: protein complexes from HEK293T cells expressing Myc-tagged KCNQ2 subunits were immunoprecipitated with an anti-Myc antibody, separated by SDS-PAGE, and analyzed by Western blotting. The arrows indicate KCNQ2 subunits and immunoprecipitated endogenous CaM detected with anti-Myc and anti-CaM antibodies, respectively. The densitometric quantification of CaM associated with the R353G mutant gave a value of ~0.6, expressed as the ratio of the optical density of immunoprecipitated CaM/KCNQ2R353G normalized to the CaM/WT-KCNQ2 value. Bottom: coimmunoprecipitation of HA-tagged KCNQ3 assembled with Myc-tagged KCNQ2 and endogenous CaM. Myc-tagged constructs were immunoprecipitated from HEK293T cells with the anti-Myc antibody, and the interacting proteins were separated by SDS-PAGE and detected by Western blotting using anti-CaM and anti-Myc antibodies. The arrowhead denotes the proteins indicated. The immunoprecipitated CaM/mutant KCNQ2 ratios normalized to the CaM/WT-KCNQ2 value for the different groups were: KCNQ2I340E + Q3: ~0.4, KCNQ2A343D + Q3: ~0.4, and KCNQ2R353G + Q3: ~0.8.

We assessed the functional consequences of the mutations that interfered with CaM binding (Fig. 2 ) by coexpressing them with KCNQ3 subunits in Xenopus oocytes. While the expression of KCNQ2 or KCNQ3 subunits alone produce only modest currents, there is a remarkable 10-fold increase when both subunits are expressed together, facilitating the functional analysis (1) . Deletion of helix A or B in KCNQ2 and point mutations that perturb CaM binding reduced the current from these channels to background levels. Furthermore, the BFNC causing mutation produced a strong reduction in current (~80%; Fig. 2 ). The current recorded could originate from homomeric KCNQ3, homomeric KCNQ2, heteromeric KCNQ2/KCNQ3, or any combination of these different populations of channels. The composition of these channels defines their sensitivity to the potassium channel blocker TEA (1 , 22) . The currents obtained from expressing KCNQ3/KCNQ2{Delta}helix A, KCNQ3/KCNQ2{Delta}helix B, KCNQ3/KCNQ2I340E, and KCNQ3/KCNQ2A343D subunits displayed weak sensitivity to TEA, comparable to that of homomeric KCNQ3 channels. In contrast, KCNQ3/KCNQ2R353G currents were TEA sensitive, a signature indicating that the residual current flowed through heteromeric channels (triangles in Supplemental Fig. 2 ). Hence, the reduction in ion current was correlated with the reduction of CaM binding to KCNQ2. Indeed, when CaM binding was more severely impaired, the channels rendered were nonfunctional (I340E, A343D); however, channels were partially functional when the decrease in CaM binding was less pronounced (R353G).


Figure 2
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  		Figure 2. CaM-binding-impaired KCNQ2 mutants do not reach the plasma membrane. A) Representative current recordings from Xenopus oocytes injected in a 1:1 ratio with cRNAs for KCNQ3 and WT-KCNQ2, or either the I340E or R353G mutant KCNQ2 subunits that disrupt CaM binding. Inset: imposed voltage protocol. B) Normalized average current and surface expression values in Xenopus oocytes of WT-KCNQ2/KCNQ3 heteromeric channels and mutant KCNQ2/KCNQ3 channels. Left panel: schematic illustration of the C-terminal region of the KCNQ2 deletions ({Delta}helix A L339-T358, {Delta}helix B S511-S523) and point mutants (I340E, A343D, R353G) analyzed. Boxes represent helices A and B; the shaded boxes indicate the CaM binding site; and the circles, the point mutations. Right panel: normalized average maximal current (gray boxes) of different KCNQ2 mutants coexpressed with KCNQ3 (n≥5) and the relative surface expression levels of KCNQ3-HA coexpressed with the same constructs (hatched boxes, n≥11). The maximal current was obtained after fitting a Boltzmann distribution to I-V relationships from tail currents measured at –20 mV after voltage prepulses from –120 mV to +50 mV. When possible, the TEA sensitivity of the currents was evaluated. Only WT-KCNQ2 and the BFNC mutant expressed heteromeric currents, as indicated by triangles (Supplemental Data). The number of KCNQ3-HA containing channels in the oocyte membrane was quantified using a whole-oocyte chemiluminiscence assay (Materials and Methods). The background of uninjected oocytes was subtracted, and the values given are the means ± SE normalized to values obtained from WT-KCNQ2/KCNQ3 (ionic currents) or WT-KCNQ2/KCNQ3-HA (chemiluminiscence) channels from the same batch.

A simple explanation for these results is that there were fewer channels at the membrane, although it has been suggested that CaM is not required for the surface expression of mouse homomeric KCNQ2 channels (12) . We re-examined this issue using a sensitive chemiluminescent assay (22 , 25) in which human KCNQ3 subunits were tagged with an HA epitope located in an extracellular loop and coexpressed with WT and mutant KCNQ2 subunits. Surface expression was greater when WT-KCNQ2 and WT-KCNQ3 were coexpressed, a process that is dependent on the heteromerization of the intracellular C-terminal region (22 , 25) . However, the number of KCNQ3 subunits at the plasma membrane did not increase above threshold detection levels when they were coexpressed with CaM-binding impaired KCNQ2 mutant subunits, even though weak surface expression was detected with the BFNC mutant R353G (Fig. 2B ). Thus, the reduction in current levels appears to be due to fewer channels reaching the plasma membrane, and CaM binding to KCNQ2 appears to be required for the correct trafficking of heterotetrameric KCNQ2/KCNQ3 channels.

Channel trafficking is a complex process that involves subunit assembly, ER exit, transport to the membrane and maintenance therein, endocytosis, and degradation (26) . One of the earliest steps is the assembly of the subunits in the ER, thus we adopted different approaches to assay the coassembly of KCNQ2 with KCNQ3. Mutant KCNQ2 channels were coexpressed with a KCNQ3 subunit carrying the A315T mutation in the inner vestibule (KCNQ3*), which, unlike WT-KCNQ3, produces robust currents when expressed alone (22) . When KCNQ3* was coexpressed with the CaM-binding deficient mutant KCNQ2 subunits or with WT-KCNQ2 subunits, a dominant negative effect on current levels was observed, indicating that these subunits coassembled (Fig. 3 A). Moreover, the residual currents displayed high TEA sensitivity when KCNQ3* was coexpressed with the WT and R353G mutant, indicating that the heteromers reached the membrane and were indeed functional. In contrast, the residual current obtained by expression of the mutants that completely abolished CaM binding to KCNQ2 (I340E, A343D, {Delta}helix A, and {Delta}helix B) was only weakly sensitive (e.g., KCNQ3* homomers), indicating that the heteromers formed were not functional. In addition, similar levels of KCNQ3 were pulled down, with the different mutant KCNQ2 channels expressed in HEK293T cells (Fig. 3B ). Additional tests to evaluate the role of CaM binding in assembly were performed on the basis that helices CD are important in this process (the SID domain) (27) . Both the full WT and the CaM-binding deficient KCNQ2I340E subunits were immunoprecipitated with a construct comprised only of helices CD (CD-TAP) and vice versa (Fig. 3C ). While it has been suggested that CaM binding might be required for the assembly of KCNQ1 subunits (28 , 29) , our data and the results of others (12) indicate that CaM does not play a significant role in the dimerization of KCNQ2/KCNQ3 subunits. Indeed, helices CD appear to be sufficient for dimerization, regardless of whether CaM binds to the channel or not.


Figure 3
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  		Figure 3. CaM binding is not required for KCNQ2/KCNQ3 dimerization. A) The ability of KCNQ2 mutants to assemble with KCNQ3 was assessed by monitoring the dominant negative effect of the KCNQ2 subunits indicated on the current levels of the pore point mutant KCNQ3A315T (KCNQ3*) in Xenopus oocytes (22) . Mean ± SE tail currents obtained at –20 mV normalized to the mean value obtained from oocytes expressing KCNQ3* alone from the same batch (n≥6). The amount of KCNQ3* cRNA expressed was maintained constant in all groups. The triangles indicate the TEA-sensitive constructs, compatible with the presence of functional heteromeric KCNQ2/KCNQ3 at the membrane (Supplemenal Data). B) Coimmunoprecipitation of HA-tagged KCNQ3 assembled with Myc-tagged KCNQ2. Proteins from HEK293T cells expressing the constructs indicated were immunoprecipitated with the anti-Myc antibody, separated by SDS-PAGE, and analyzed in Western blots probed with anti-HA or anti-Myc antibodies. The arrows point to the proteins indicated. C) Coassembly of the TAP-tagged assembly domain construct (CD-TAP) with the Myc-tagged KCNQ2 subunit or with the CaM-binding incompetent KCNQ2I340E mutant. The TAP epitope comprises 2 affinity sequences, an IgG-binding domain from protein A and the CaM-binding peptide (CBP). HEK293T cells expressing CD-TAP with KCNQ2 or KCNQ2I340E were solubilized and immunoprecipitated with either an anti-Myc antibody (left panel) or IgG-sepharose (right panel), and the proteins were separated by SDS-PAGE and analyzed in Western blots probed with anti-CBP or anti-Myc antibodies. The asterisk labels the heavy chain of the mouse primary antibody that is recognized by the secondary antibody.

To monitor the intracellular distribution of the channels, KCNQ2 subunits were tagged at the N terminus with the blue-emitting fluorescent protein mCFP (rendered green), and these subunits were coexpressed with a red-emitting fluorescent ER marker. This N-terminal tag did not significantly affect the electrophysiological properties of the channels expressed in Xenopus oocytes (not shown). Conventional colocalization studies are based on a dye-overlay method, such that the superimposition of green and red images gives a yellow color (Fig. 4 A). To compare the degree of colocalization, 32 confocal images were acquired simultaneously at 32 different wavelengths, and they were subjected to spectral linear unmixing (permitting the emission of one fluorophore to be separated from another), followed by a pixel-to-pixel comparison (23) .


Figure 4
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  		Figure 4. Ca2+-CaM regulates the traffic of KCNQ2 subunits. A) Effect of overexpressing CaM and the Ca2+-binding mutant CaM1234 on the distribution of KCNQ2 channels. Left panel: confocal images of HEK293T cells cotransfected with mCFP-tagged WT and mutant KCNQ2 subunits, and with a DsRed-tagged ER marker. The cells were processed as described in Material and Methods. The KCNQ2 subunits and the ER marker were pseudocolored in green and red, respectively, resulting in a yellow color when they colocalized. Center panel: the effect of elevated CaM expression. Right panel: the effect of elevating the expression of a Ca2+ binding incompetent CaM mutant (CaM1234). Scale bar = 10 µm. The bars on the right represent averaged Manders coefficient of colocalization of cells expressing the different constructs indicated at left (n≥10). Error bars, SEM; *P < 0.05; **P < 0.001; unpaired t test. The contribution of the intracellular signals to this index is much more important than the signals from the plasma membrane, when present. For instance, the index obtained for the Tac-CFP construct (Fig. 5 ; Supplemental Data) that clearly decorates the membrane was around 0.56. Thus, the expected range in our working conditions was from 0.56 for a membrane protein to 1.0 for a protein fully retained at the ER. B) Effect of sequestering CaM on the distribution of KCNQ2 subunits. Left panel: confocal images of mCFP-tagged WT-KCNQ2 with elevated expression of the indicated CaM-buffering constructs processed as explained in Results. Scale bar = 10 µm. Right panel: averaged Manders coefficient for colocalization in cells overexpressing the CaM-buffering proteins indicated at left (n≥8). Error bars, SEM; *P < 0.05; **P < 0.001; unpaired t test.

While a fraction of the KCNQ2 signal colocalized with the ER marker, another part was found in punctate structures within the cell. The fraction of channels in the ER was clearly greater when both the I340E mutant and the BFNC causing mutant R353G were analyzed, suggesting that CaM binding is required for the channels to exit the ER. No major alteration in the distribution of WT-KCNQ2 subunits was detected after overexpression of CaM. Likewise, the distribution of the reference I340E mutant was not affected by incrementing the levels of CaM, reflecting the inability of this mutant to bind CaM. However, the distribution of the BFNC causing mutant was altered (Fig. 4A ) in accordance with the partial impact of this mutation on CaM binding. Indeed, the fraction of WT channels retained in the ER increased when the amount of free CaM fell in the cells because of the elevated expression of 2 different CaM binding proteins, neurogranin and helices AB from KCNQ2 (Fig. 4B ). In contrast, a construct carrying the I340A mutation that binds much less CaM (11) had a weaker effect on channel distribution, further evidence of the involvement of helices AB from KCNQ2 in sequestering CaM. In addition, sequestering CaM with neurogranin did not alter the distribution of Tac-CFP, an unrelated membrane protein that does not bind CaM (Supplemental Data).

The impact of Ca2+ binding to CaM on KCNQ2 trafficking was evaluated using the CaM1234 mutant that is unable to bind Ca2+ but which does associate with KCNQ2 subunits (12) . In contrast to the effect of CaM, overexpression of this CaM mutant provoked the retention of more WT-KCNQ2 channels in the ER while exerting little or no effect on the distribution of the R353G or the reference I340E mutant channels (Fig. 4A ). These results suggested that the channels might still exit the ER when Ca2+-CaM was replaced by CaM1234, albeit less efficiently. Indeed, functional channels can be recorded from cells overexpressing this mutant CaM (12 , 13) . Hence, the full effect of the overexpression of CaM1234 will be complex. On one hand, there will be a tendency to reduce the number of channels at the plasma membrane while, on the other, the channels that reach the membrane will not be subjected to Ca2+-dependent inhibition, permitting larger current flow (13) . Thus, the net effect on current amplitude will reflect the balance between reduced surface expression and the release from Ca2+-dependent inhibition.

The impact of CaM binding to helices AB might be dependent on other regions of the channel. To explore this possibility, we fused helices AB to the intracellular C terminus of the single transmembrane interleukin-2 receptor {alpha} subunit (Tac; Fig. 5 A). Tac harbors 2 N-glycosylation sites that are processed in the Golgi apparatus, thereby producing more complex forms with substantial differences in molecular weight (30 , 31) . When helices AB from KCNQ2 were inserted into the Tac-CFP chimera, a high molecular weight band was observed, which corresponded to the glycosylated isoform. The intensity of this band decreased when the constructs carried the I340E or the R353G mutations that abolished or reduced CaM binding (Fig. 5B ). Moreover, glycosylation of the WT increased after CaM overexpression, but glycosylation of the I340E mutant remained unaltered. Indeed, the overexpression of the Ca2+-independent CaM mutant (CaM1234) did not augment glycosylation, further reinforcing the role of Ca2+.


Figure 5
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  		Figure 5. Effect of helices AB on the maturation of Tac fusion proteins. A) Schematic representation of the Tac-Q2AB-CFP construct. Two positions for the addition of N-linked oligosaccharide chains are indicated (CHO). CFP was fused to the intracellular C-terminal domain of the Tac receptor and the CaM binding domain was inserted between the Tac-CFP construction. B) Western blot probed with an anti-GFP antibody of HEK293T cell extracts expressing either Tac-CFP, Tac-Q2AB-CFP, Tac-Q2ABI340E-CFP, or Tac-Q2ABR353G-CFP and, when indicated, with YFP-CaM, YFP-CaM1234, or YFP-Neurogranin that were overexpressed and which also are recognized by the anti-GFP antibody used (arrow). The top bands represent immature (*) and mature (**) forms of Tac chimeras. The mature band indicates a complex glycosylation state acquired after processing in the Golgi system (40) . Bottom: The averaged ratio of optical density values (immature OD/mature OD) normalized to the Tac-AB ratio (n≥3) and compared to the normalized ratio for Tac-GFP of ~260. C) Confocal images of cells coexpressing WT Tac-AB-CFP (a) or the CaM-binding deficient I340E mutant (c; rendered in green), an ER marker (rendered in red), and YFP-CaM (not shown in a, c). YFP-CaM filled the cell (insets; b, d) allowing the filopodia-like structures to be seen (arrows). The insets are the upper confocal planes of the cells (a and c show the signal from Tac-chimeras, b and d from YFP-CaM), highlighting that WT Tac-AB-CFP decorated the filopodia (note the similar pattern of insets in a, b), unlike the I340E mutant (note the contrasting pattern of insets in c, d).

In contrast to the KCNQ2 subunits, the levels of endogenous CaM appeared to be limiting for Tac-AB, perhaps since its affinity for CaM may be lower than that of KCNQ2. (Presumably, we are comparing a monomeric protein with a tetrameric protein.) Overexpression of WT-CaM did not modify the subcellular location of KCNQ2 nor did it increase its exit from the ER (Fig. 4A ). In contrast, the same experimental manipulation increased the glycosylation of the Tac-AB protein (Fig. 5B ), suggesting that it exits the ER more readily when CaM is bound. Conversely, while coexpression of KCNQ2 subunits and CaM1234 increased ER retention (Fig. 4A ), this mutant CaM did not modify the processing of the Tac-AB protein (Fig. 5B ). Although the effects of CaM overexpression on KCNQ2 subunits and Tac-AB constructs were different, they support the idea that CaM is required to exit the ER and that Ca2+-CaM is more effective than apo-CaM in promoting trafficking.

The membrane regions labeled in the images obtained from cells expressing these Tac constructs presented a strikingly different pattern. The cells displayed numerous filopodia-like protrusions of the plasma membrane that were decorated in cells expressing the WT helix AB construct but not in those cells expressing the I340E mutant. This pattern was clearest in cells overexpressing CaM, showing that more protein reached the plasma membrane and that CaM binding was a requirement for trafficking. Thus, the helices AB from KCNQ2 harbor molecular determinants required for the Ca2+-CaM regulation of trafficking that can be transplanted to an unrelated protein, indicating that this process is independent of other regions of the channel.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is remarkable that, although KCNQ2 is functional in the plasma membrane, the majority of this protein is located in the ER and other intracellular compartments, both in neurons and in cells heterologously expressing the channel (32 , 33) . Indeed, sophisticated amplification procedures generally are required to detect the channel at the cell surface (33) . This means also that there is a large reservoir of intracellular channels that may traffic to the plasma membrane under the appropriate conditions, even though a membrane protein has to overcome multiple quality control mechanisms to reach the plasma membrane. The results presented here indicate that Ca2+-CaM binding is required for KCNQ2 channels to get through the trafficking checkpoints at the ER "exit sites," although it is not necessary at earlier stages, such as for subunit dimerization. In fact, sequestering CaM or the introduction of mutations into the channel that impair CaM binding led to retention in the ER compartment. In addition, CaM overexpression facilitated the exit from the ER of a mutant channel associated with BFNC epilepsy that partially retained the ability to interact with CaM. Moreover, CaM seems to confer Ca2+ sensitivity to the membrane trafficking of KCNQ2 channels, given that overexpression of a Ca2+-binding incompetent CaM mutant led to retention of the channels and did not promote glycosylation of the Tac trafficking reporter. Although the experiments indicate that the effect of CaM is solely dependent on helices AB, further studies will be required to ascertain whether the role of CaM is context or position sensitive. Considering the number of membrane proteins that also bind CaM and display a similar subcellular distribution to KCNQ2, this mechanism may represent a widespread checkpoint for controlling local or general membrane insertion.

Our results do not support the conclusion drawn from earlier studies (12) . While it was previously concluded that the surface expression of CaM-binding deficient KCNQ2 mutant channels was "more or less normal," quantitative differences were noted in membrane targeting consistent with the results presented here (12) . Species-specific differences (human vs. mouse) and the use of different splice variants (human isoform 3 vs. mouse isoform 1) may account for some of the differences observed.

Local levels of Ca2+ and CaM may have profound effects on the membrane expression of M-channels. Certainly, reducing the availability of free CaM in hippocampal neurons decreases M-current density and increases membrane excitability. These effects are observed without affecting A-type or large conductance Ca2+-activated AHP-type K+ channels (19) . Free CaM levels are controlled by CaM-binding proteins, such as GAP43 (neuromodulin) and neurogranin (34) . Interestingly, we found that elevating the levels of neurogranin disrupts KCNQ2 channel trafficking. Given that neurogranin concentrates in dendritic spines (35 , 36) , KCNQ channels may preferentially insert into the membranes in compartments with less neurogranin, such as the soma and axons. Indeed, KCNQ channels are preferentially localized at the surface of axons, both in the initial axon segment and in more distal domains (8) . While targeting of KCNQ channels to the surface of the initial axon segment is mediated by ankyrin-G binding motifs, sequences mediating targeting to the surface of more distal portion of the axon reside in different regions of the C-terminus, including the membrane proximal CaM-binding site (8) . It is plausible that local activity-dependent increases in the Ca2+ concentration at distal regions of the axon could be decoded by the Ca2+-CaM machinery described here. Accordingly, this action would drive the trafficking of the channels to discrete regions of the plasma membrane and, hence, produce changes in synaptic activity.

In many cases, CaM binds constitutively to proteins in the absence of Ca2+, and Ca2+ binding triggers a conformational change that modulates their function (see, for instance, refs. 37 , 38 ). This arrangement ensures a fast response to Ca2+ oscillations (in the millisecond time scale), although it is unlikely that such velocity is physiologically relevant for a process as slow as membrane protein trafficking. Binding in the absence of Ca2+ may be required to increase the local concentrations of CaM in the surroundings of the protein, thereby enhancing the efficacy of the response (39) . Alternatively, this Ca2+-independent binding may be more relevant for other purposes, such as Ca2+-mediated inhibition (13) .

While binding to apo-CaM simultaneously requires helices A and B, Ca2+-CaM can bind to either helix in vitro (11 12 13) . Which helix relays the information to trigger the Ca2+-CaM mediated release of the channels from the ER remains to be elucidated. Both helix A and helix B have clusters of lysine and arginine residues, an organization that is reminiscent of certain retention/retrieval signals. When exposed, these signals are recognized by the quality control machinery of the ER, impairing the exit of the protein to forward organelles. One attractive hypothesis is that Ca2+-CaM may mask such a signal. Helices AB share the "transplantation capacity" of retention/retrieval signals, sequences that are sufficient to provoke protein retention even in an unnatural context. The glycosylation state of Tac chimeras allowed us to monitor their transit into the Golgi apparatus, and the results obtained are consistent with a role of Ca2+-CaM in masking a retention signal. Indeed, glycosylation of Tac-AB-CFP was almost completely suppressed when CaM binding was disrupted, and it increased as the levels of CaM rose. Whether the features that helices AB share with retention/retrieval signals correspond to discrete residues or are dispersed along the sequence remains to be seen. Likewise, the cellular machinery that recognizes those features remains to be identified.

In summary, we have identified a mechanism that controls the membrane expression of KCNQ2 channel subunits and that could explain the pathogenesis of a mutation responsible for a type of human epilepsy. This mechanism may be used by many membrane proteins to control their expression at the cell surface. Further experiments will be required to determine the full implications of this Ca2+ sensitive trafficking checkpoint.


   ACKNOWLEDGMENTS
 
We thank Rosa Mella, Almudena Areso, Mark Sefton, and Irene Santana Castro for their contributions. P.V. and A.A. hold fellowships from the Basque Government, J.C.G.P. holds an I3P CSIC fellowship, and P.A. holds a fellowship from the Spanish Ministry of Education. This work was supported by grants from the VI European framework program, Spanish Ministry of Education, and Bizkaiko Foru Aldundia.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication August 24, 2007. Accepted for publication October 11, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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