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Research Report
The Kv1.2 potassium channel: The position of an N-glycanon the extracellular linkers affects its protein expressionand function
Jing Zhua, Esperanza Recio-Pintob, Torsten Hartwiga, Will Sellersa,Jingyi Yanc, William B. Thornhilla,⁎aDepartment of Biological Sciences, Fordham University, Bronx, NY 10458, USAbDepartment of Anesthesiology, New York University Langone Medical Center, New York, NY 10016, USAcThe Bronx High School of Science, Bronx, NY 10468, USA
Article history:Accepted 10 November 2008Available online 20 November 2008
Voltage-gated potassium Kv1 channels have three extracellular linkers, the S1–S2, the S3–S4, and the S5-P. The S1–S2 is the only linker that has an N-glycan and it is at a conservedposition on this linker on Kv1.1–Kv1.5 and Kv1.7 channels. We hypothesize that an N-glycanis found at only this position due to its effect on folding, trafficking, and/or function of thesechannels. To investigate this hypothesis, N-glycosylation sites were engineered at differentpositions on the extracellular linkers of Kv1.2 to determine the effects of N-glycans onchannel surface protein expression and function. Our data suggest that for Kv1 channels, (1)placing an N-glycan at non-native positions on the S1–S2 linker decreased cell surfaceprotein expression but the N-glycan still affected function similarly as if it were at its nativeposition, (2) placing a non-native N-glycan on the S3–S4 linker significantly altered function,and (3) placing a non-native N-glycan on the S5-P linker disrupted both trafficking andfunction. We suggest that Kv1 channels have an N-glycan at a conserved position on onlythe S1–S2 linker to overcome the constraints for proper folding, trafficking, and function thatappear to occur if the N-glycan is moved from this position.
Voltage-gated potassium (Kv) channels are membrane pro-teins that control the excitability of nerve and muscle bymodulating the resting membrane potential and conditioningthe shape and frequency of action potentials (Hodkgin andHuxley, 1952;Hille, 2001). During the biogenesis of Kv chan-nels, four α subunits assemble to form an ion conduction pore,
.B. Thornhill).channel; K+, potassium
t cells; Endo H, endoglycoalf activation; G–V curve
er B.V. All rights reserved
which is essential for potassium ion (K+) selectivity andpermeation (Coleman et al., 1999; Dodson et al., 2003).Auxiliary β subunits can associate with α subunits to modifyfunction of the channel complex (Rhodes et al., 1995).
A Kv α subunit contains a highly conserved core region,consistingof six transmembranedomains,whichare connectedby intracellular and extracellular linkers, and variable cytoplas-mic amino and carboxyl termini (Coetzee et al., 1999). Many Kv
ion; ER, endoplasmic reticulum; CHO, Chinese hamster ovary cells;sidase H; PNGase F, peptide N-glycosidase F; NXT, N-glycosylation, normalized conductance–voltage curve
Fig. 1 – A schematic of the S1–S2 linker of Kv1 channelN-glycosylation at different positions. (A) A diagram of themembrane topology of a monomeric Kv1 subunit. S1 to S6denote six transmembrane domains and P denotes the poreregion. Also shown are three extracellular linkers: S1–S2,S3–S4, and S5-P linkers. The NXT indicates theN-glycosylation site. (B–C) S1–S2 linker amino acid sequencesof Kv1.2 (B) and Kv1.4 (C). The N-glycosylation accessibleregion (see text for definition) is underlined. The numbersabove the S1–S2 sequence correspond to the position ofamino acids in the context of the linker; and the numbersbelow the S1–S2 sequence represent the position of aminoacids in the context of the full length protein. N-glycosylationsites were engineered to Kv1 constructs at position #13 or#19. The numbers with asterisks indicate nativeN-glycosylation sites that are glycosylated.
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channels are N-glycosylated on their extracellular linkers(Stuhmer et al., 1989). N-glycosylation begins with the cotran-slational addition of a core glycan to a nascent polypeptide inthe endoplasmic reticulum (ER), and the glycan is furtherprocessed in the Golgi apparatus (Varki, 1998;Yoshida, 2003).N-glycosylation promotes proper folding and assembly,increases stability and trafficking, and/or modifies the functionof a variety of K+ channels (Thornhill et al., 1996; Petrecca et al.,1999; Freeman et al., 2000; Khanna et al., 2001; Watanabe et al.,2003;Napp et al., 2005; Sutachan et al., 2005; Hagen andSanders,2006; Watanabe et al., 2007; Johnson and Bennett, 2008) andother membrane proteins (Covarrubias et al., 1989; Recio-Pintoet al., 1990; Gurnett et al., 1996; Bennett et al., 1997;Ufret-Vincentyet al., 2001; Bennett, 2002). Kv1.1–Kv1.5 and Kv1.7 channels areN-glycosylated on the first extracellular linker between trans-membrane domains S1 and S2 (the S1–S2 linker). Kv1.1 or Kv1.2without their N-glycans exhibited altered functional properties(Thornhill et al., 1996; Watanabe et al., 2003, 2007), and Kv1.4without its N-glycan showed a decreased protein stability andtrafficking to the cell surface (Watanabe et al., 2004). Kv3channels contain two native N-glycosylation sites in the S1–S2linker. Occupancy of these two sites byN-glycans affects foldingandmaturation of functional Kv3.1 at the cell surface (Brooks etal., 2006). Kv10.1 is N-glycosylated in the turret region betweenthe S5 transmembrane domain and the pore domain (the S5-Plinker). A complex type N-glycan in this region is required forproper trafficking and correct function of Kv10.1 (Napp et al.,2005). However, the effects on protein stability, trafficking, andfunction of moving an N-glycan to different positions on anextracellular linker of an ion channel have not been tested.
Our previousworkhas shown that themiddle section of theS1–S2 linker of Kv1.2 was accessible to glycotransferases, andevery position in this section could be N-glycosylated when anN-glycosylation sitewas engineered here (Zhu et al., 2003). Themiddle section was referred to as the accessible region. Asimilar accessible region was also found in the S1–S2 linker ofKv1.4 (unpublished data), suggesting similar secondary struc-tures of the S1–S2 linkerswithin the Kv1 channels. However, inspite of a wide accessible region, the native N-glycans wereonly attached to ahighly conservedposition in the S1–S2 linkerof the Kv1 channels. Additionally, the other extracellularlinkers, the S3–S4 and S5-P linkers, of Kv1 channels do nothave any N-glycans. We hypothesize that the conservation ofanN-glycan position on the S1–S2 linker of Kv1 channels is dueto structural and/or functional restrictions.
In this study, we investigated (1) whether the position of anN-glycan on the S1–S2 linker affects stability, maturation, andtrafficking as well as functional properties of Kv1.2; and (2) theeffects of placing an N-glycan on the other extracellularlinkers on Kv1.2.
2. Results
2.1. The position of an N-glycan on the S1–S2 linker ofKv1.2 altered cell surface protein levels but not the channel'sfunctional properties
Kv1.2 has one N-glycosylation consensus site (NST) on its S1–S2 linker at N207 (Figs. 1A and B) and this site is glycosylated
(Zhu et al., 2003). To address whether there is any positionaleffects of an N-glycan, Kv1.2N207Q, which is Kv1.2 with itsnative N-glycosylation sitemutated, was used as a template toengineer a NST site at position #13 or #19 (these numbers referto amino acid numbers in the context of only the S1–S2 linker)(Fig. 1B), and thus, there is only oneN-glycosylation consensussite on the extracellular linkers on these constructs. In thecontext of the full length protein the amino acid numbers are195 or 201 for #13 or #19, respectively (Fig. 1B). Position #13 and#19 are located at the N-terminal end and the middle sectionof the accessible region of the S1–S2 linker, respectively. Thenative N-glycosylation site of Kv1.2 is at position #25, or 207 inthe context of full length, which is on the C-terminal end ofthe accessible region. Kv1.2 and mutant proteins weretransiently expressed in CHO cells, which do not endogen-ously express Kv1 subfamily member proteins (Helms et al.,1997) or Kvβ subunits (Shamotienko et al., 1999), and thus, thechannel would be expressed as a homotetramer.
On immunoblots, Kv1.2, Kv1.2#13, and Kv1.2#19 showed twodistinct bands with electrophoretic mobilities corresponding to
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~60 and 80 kDa, respectively (Fig. 2A). The 60 kDa band wassensitive to both EndoH and PNGase F, suggesting that itcontains a high-mannose type N-glycan, and was referred toas an immature protein. In contrast, the 80 kDa band wassensitive to PNGase F, but resistant to Endo H, indicating that
it contains a complex type N-glycan, and was referred to as amature protein. The upper band in lanes 3, 6, and 9 afterPNGase F treatment is a phosphorylated band because it issensitive to phosphatase treatment (data not shown). The cellsurface protein levels of Kv1.2 constructs were estimated bybiotinylation and immunoblotting. Kv1.2#13 and Kv1.2#19showed significantly decreased surface protein levels, whichwere only ~28% the level of Kv1.2 wild type (Fig. 2B). Inaddition, cell surface functional expression levels wereestimated by whole cell patch-clamping and these twomutants exhibited only ~40% the conductance density asKv1.2 (Fig. 2E).
The decrease in surface protein level could be caused byeither a decreased protein stability or a decreased proteintrafficking. To address this question, the total protein levels ofKv1.2 constructs weremeasured by immunoblotting. Kv1.2#13and Kv1.2#19 showed similar total protein levels comparedwith Kv1.2 (Fig. 2C), suggesting that the two mutants hadsimilar protein stabilities compared with wild type. Dividingthe surface protein (Fig. 2B) by the total protein (Fig. 2C) givesthe relative surface expression level (Fig. 2D), which can be
Fig. 2 – Impact on cell surface protein levels when anN-glycan is attached to different positions on the S1–S2 linkerof Kv1.2. Kv1.2 constructs were transiently expressed in CHOcells. The surface protein levels were estimated bybiotinylation/immunoblotting and the total protein levelswere estimated by immunoblotting. (A) Endo H and PNGase Ftreatments of Kv1.2 proteins. The number to the left of thefigure denotes the electrophoretic mobility in kDa. Themature band indicates proteins containing complex typeN-glycans that are sensitive to PNGase F and resistant toEndo H. The immature band indicates proteins containinghigh-mannose type N-glycans that are sensitive to both EndoH and PNGase F. (B) Immunoblot analysis of surface proteins.The means of Kv1.2#13 and Kv1.2#19 were normalized toKv1.2, which was taken as 100.0±SE, n=3. The surfaceprotein levels of Kv1.2#13 and Kv1.2#19 are statisticallydifferent from Kv1.2 (p<0.05). (C) Immunoblot analysis oftotal proteins. The means of Kv1.2#13 and Kv1.2#19 werenormalized to Kv1.2, whichwas taken as 100.0±SE, n=3. Thetotal protein levels of all three constructs are not statisticallydifferent (p>0.05). (D) Relative surface expression estimatedby dividing surface protein (B) by total protein (C). The relativesurface expression of Kv1.2#13 and Kv1.2#19 are statisticallydifferent from Kv1.2 (p<0.05). (E) Conductance density wasestimated by whole cell patch-clamping as described inExperimental procedures. Kv1.2 values were set to 100.0±SEand the other mutant values were normalized to it, n=7–10.The conductance densities of Kv1.2#13 and Kv1.2#19 arestatistically different from Kv1.2 (p<0.05). (F–H) Surfaceprotein level (F), total protein level (G), and relative surfaceexpression (H) of Kv1.2 constructs were measured whenexpressed in CHO cells in the presence of tunicamycin(2 μg/ml) which inhibits N-glycosylation (n=3). There are nostatistical differences in surface protein level, total proteinlevel, and relative surface expression among all threeconstructs (p>0.05). Statistical analysis was by ANOVA andTukey's multiple comparison tests.
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considered a trafficking efficiency index versus a control (inthis case Kv1.2 wild type). An index number less than one for aconstruct suggests that it was intracellularly retained com-pared with the control. Kv1.2#13 and Kv1.2#19 showeddecreased relative surface expression compared with Kv1.2,indicating that these two mutants had decreased traffickingefficiency compared withwild type. These results suggest thatKv1.2#13 and Kv1.2#19 had decreased surface protein levelsbecause they both had decreased trafficking efficiency.
To determine whether the reduction in surface proteinlevel of Kv1.2 mutants is due to the positional effects of N-glycans or the replacement of three amino acids at the N-glycosylation site, CHO cells were transfected and incubated intunicamycin, an antibiotic that inhibits N-glycosylation. In thepresence of tunicamycin, all Kv1.2 constructs had similarsurface protein level, total protein level, and relative surfaceexpression (Figs. 2F–H). The upper band in all lanes in Fig. 2F isa phosphorylated protein, as discussed previously. We alsorecorded similar results as with tunicamycin using trans-fected CHO glycosylation-deficient Lec1 cells, which produceonly high-mannose type N-glycans on glycoproteins (Stanleyand Siminovitch, 1977, data not shown). These findingssuggest that it was the placement of an N-glycan at variouspositions on the S1–S2 linker that was responsible for theresults here and not amino acid replacements per se.
The above results indicate that attaching an N-glycan tothe N-terminal end (#13) or to the middle section (#19) of theaccessible region decreased Kv1.2 surface expression byinducing intracellular retention. Thus, an N-glycan at itsnative position in the S1–S2 linker, the C-terminal end of theaccessible region, was required for wild type protein traffick-ing to the cell surface.
Functional parameters were further recorded for Kv1.2constructs by whole cell patch-clamping. Kv1.2#13 andKv1.2#19 showed similar activation kinetics, voltage depen-
Fig. 3 – Impact on functional properties when an N-glycan is att(A) Scaled whole cell currents from Kv1.2, Kv1.2#13, and Kv1.2#1decreased to obtain somewhat similar size peak currents as Kv1activation kinetics may be viewed directly. Peak currents beforeKv1.2#13, and Kv1.2#19, respectively. Cells were held at −80 mV(B) Table of voltage dependence of half activation (V1/2) values andnot shown) for Kv1.2 constructs in CHO cells (mean±SE, n=8–15)significantly different, as analyzed by one-way ANOVA and Tuk
dence of half activation (V1/2), and slopes of normalizedconductance–voltage (G–V) curves compared with Kv1.2 wildtype in CHO cells (Figs. 3A and B). Furthermore, Kv1.2constructs were transfected into glycosylation-deficient Lec1cells to control for amino acid replacements at the N-glycosylation site. As expected, all parameters examinedwere similar for Kv1.2 mutants and wild type (data notshown). It appears that moving an N-glycan to differentpositionson theKv1.2 S1–S2 linker affected thevoltage-sensingmachinery similarly as when an N-glycan was at its nativeposition.
2.2. Kv1.4 exhibited similar positional effects of anN-glycan on its S1–S2 linker
To address whether our findings for Kv1.2 were predictive foranother Kv1 member, similar experiments were applied toKv1.4. Kv1.4 has one N-glycosylation consensus site (NDT) onits S1–S2 linker at N354 (Figs. 1A and C) and this site isglycosylated (Watanabe et al., 2004). Kv1.4N354Q, which isKv1.4 with its native N-glycosylation site mutated, wasengineered to contain a NDT site at positions #13 or #19 onits S1–S2 linker (Fig. 1C) and thus, there is only one N-glycosylation site on these constructs—numbers refer toamino acid numbers only in the context of the S1–S2 linker.In the context of the full length protein the amino acidnumbers are 341 or 347 for #13 or #19, respectively. These twopositions were chosen to match the positions tested in theKv1.2 S1–S2 linker.
On immunoblots, Kv1.4, Kv1.4#13, and Kv1.4#19 showedtwo distinct bandswith electrophoreticmobilities correspond-ing to ~85 and 110 kDa, respectively (Fig. 4A). The 85 kDa bandwas sensitive to both EndoH and PNGase F, suggesting that itcontains high-mannose type N-glycan, and was referred to asan immature protein. In contrast, the 110 kDa band was
ached to different positions on the S1–S2 linker of Kv1.2.9 transfected CHO cells. The cDNA amount of Kv1.2 was.2 mutants. Traces were then scaled so that differences inscaling at 50 mV were 2.2 nA, 3.1 nA, and 3.1 nA for Kv1.2,and depolarized to 50 mV in 10 mV increments for 80 ms.slopes of normalized conductance–voltage (G–V ) curves (data. The V1/2 values and slopes of Kv1.2 constructs are notey's multiple comparison tests (p>0.05).
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sensitive to PNGase F, but resistant to EndoH, indicating that itcontains a complex type N-glycan, and was referred to as amature protein (Fig. 4A). The cell surface protein levels ofKv1.4#13 and Kv1.4#19 decreased significantly and were only~50% the level of Kv1.4 wild type (Fig. 4B). A similar result wasobtained with Kv1.4 mutants when conductance density wasused to estimate surface expression levels (Fig. 4E). Next, the
total protein levels were measured. The total protein level ofKv1.4#13 was decreased, but the total protein level of Kv1.4#19was similar to Kv1.4 (Fig. 4C). Dividing the surface protein(Fig. 4B) by the total protein (Fig. 4C) gives the relative surfaceexpression level (Fig. 4D). Kv1.4#13 showed a similar relativesurface expression level compared with Kv1.4, suggestingthat the mutant had a similar trafficking efficiency. On theother hand, the relative surface expression level of Kv1.4#19was decreased ~60% compared with Kv1.4, suggesting thatthe mutant had a decreased trafficking efficiency comparedwith wild type. These results suggest that the decreasedsurface protein levels of Kv1.4 mutants were caused by adecreased protein trafficking or a decreased protein stability.In the presence of tunicamycin, all Kv1.4 constructs hadsimilar surface protein levels, total protein levels, and relativesurface expression levels (Figs. 4F–H), which suggests that anN-glycan addition at different positions affected parametersand not amino acid replacements per se. Thus, similar resultson positional effects of an N-glycan at #13 or #19 on the S1–S2linker were recorded for both Kv1.2 and Kv1.4.
Fig. 4 – Impact on cell surface protein levels when anN-glycan is attached to different positions on the S1–S2 linkerof Kv1.4. Kv1.4 constructs were transiently expressed inCHO cells. The surface expression levels were estimated bybiotinylation/immunoblotting and the total protein levelswere estimated by immunoblotting. (A) Endo H and PNGase Ftreatments of Kv1.4 proteins. The number to the left of thefigure denotes the electrophoretic mobility in kDa. Themature and immature bands were defined as in Fig. 2.(B) Immunoblot analysis of surface proteins. The means ofKv1.4#13 and Kv1.4#19were normalized to Kv1.4, whichwastaken as 100.0±SE, n=3. Surface protein levels of Kv1.4#13and Kv1.4#19 are statistically different from Kv1.4 (p<0.05).(C) Immunoblot analysis of total proteins. The means ofKv1.4#13 and Kv1.4#19were normalized to Kv1.4, whichwastaken as 100.0±SE, n=3. The total protein level of Kv1.4#13 isstatistically different from Kv1.4 (p<0.05), but the totalprotein level of Kv1.4#19 is not statistically different fromKv1.4 (p>0.05). (D) Relative surface expressionwas estimatedby dividing surface protein (B) by total protein (C). The relativesurface expression level of Kv1.4#13 is not statisticallydifferent from Kv1.4 (p>0.05) but the relative surfaceexpression level of Kv1.4#19 is statistically different fromKv1.4 (p<0.05). (E) Conductance density was estimated bywhole cell patch-clamping as described in Experimentalprocedures. Kv1.4 values were set to 100.0±SE and the othermutant values were normalized to it, n=7–9. Theconductance densities of Kv1.4#13 and Kv1.4 #19 arestatistically different from Kv1.4 (p<0.05). (F–H) Surfaceprotein level (F), total protein level (G), and relative surfaceexpression (H) of Kv1.4 constructs were measured whenexpressed in CHO cells in the presence of tunicamycin(2 μg/ml) which inhibits N-glycosylation (n=3). There are nostatistical differences in surface protein level, total proteinlevel, and relative surface expression among all threeconstructs (p>0.05). Statistical analysis used ANOVA andTukey's multiple comparison tests.
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2.3. A non-native N-glycan on an extended S3–S4 linker ofKv1.2 altered channel function
The S3–S4 extracellular linker of Kv1.2 does not contain anynative N-glycosylation sites (Fig. 5A). Our previous studyshowed that when an N-glycosylation site (NST) was engi-neered to themiddle section of the S3–S4 linker of Kv1.2N207Q
(Kv1.2 with its native N-glycosylation site mutated), theconstruct, termed Kv1.2N1, was not glycosylated (Zhu et al.,2003). Presumably, the linker is too short to be glycosylatedbecause generally a site that is glycosylated is 10 amino acidsfrom a transmembrane domain (Landolt-Marticorena andReithmeier, 1994). However, Kv1.2N2, which is Kv1.2N1 withan extended S3–S4 linker (14 amino acidswere added) (Fig. 5A),was expressed as a glycoprotein (Zhu et al., 2003). A wild typevoltage-gated potassium channel has not been described thathas an N-glycan on this linker. We hypothesized that an N-glycan on this linker would be structurally or functionallyunfavorable. To test this hypothesis, the protein expressionlevels of Kv1.2N2, which contains one N-glycan on the S3–S4linker, and Kv1.2 wild type, which contains one N-glycan onthe S1–S2 linker, were measured. The surface protein level,total protein level, and relative surface expression level ofKv1.2N2 were similar to or slightly different from Kv1.2 (Figs.5B–D). In contrast, the conductance density level of Kv1.2N2was only 40% the level of Kv1.2 (Fig. 5E). It appears thatmovinganN-glycan from the S1–S2 linker to the extended S3–S4 linkerhad little or no effect on stability, trafficking, and surfaceprotein expression of Kv1.2 but it did decrease functionalconductance density levels. To control for the extended linker,
Fig. 5 – Impact on cell surface protein levels when anN-glycan is attached to the S3–S4 linker of Kv1.2. Kv1.2constructs were transiently expressed in CHO cells or Lec1cells. The surface expression levels were estimated bybiotinylation/immunoblotting and the total protein levelswere estimated by immunoblotting. Conductance densitywas estimated by whole cell patch-clamping as described inExperimental procedures. (A) S3–S4 linker sequences ofKv1.2 constructs. Engineered N-glycosylation sites areunderlined. (B) Immunoblot analysis of surface proteins fromCHO cells. The mean of Kv1.2N2 was normalized to Kv1.2,which was taken as 100.0±SE, n=3. The surface proteinlevels of Kv1.2 and Kv1.2N2 are statistically different(p<0.05). (C) Immunoblot analysis of total proteins from CHOcells. The mean of Kv1.2N2 was normalized to Kv1.2, whichwas taken as 100.0±SE, n=3. The total protein levels of Kv1.2and Kv1.2N2 are not statistically different (p>0.05). (D)Relative surface expression was estimated by dividing thesurface protein (B) by the total protein (C). The relative surfaceexpression levels of Kv1.2 and Kv1.2N2 are not statisticallydifferent (p>0.05). (E) Conductance density analysis in CHOcells. The mean of Kv1.2 was set to 100.0±SE and Kv1.2N2was normalized to it, n=7–10. The conductance densities ofKv1.2 and Kv1.2N2 are statistically different (p<0.05).(F–H) Surface protein level (F), total protein level (G), andrelative surface expression (H) of Kv1.2 and Kv1.2N2 weremeasured when expressed in CHO cells in the presence oftunicamycin (2 μg/ml) which inhibits N-glycosylation (n=3).There are no statistical differences in surface protein level,total protein level, and relative surface expression among althree constructs (p>0.05). (I) Conductance density analysis inLec1 cells. The mean of Kv1.2 was set to 100.0±SE andKv1.2N2 was normalized to it, n=12–17. The conductancedensities of Kv1.2 and Kv1.2N2 are not statistically different(p>0.05). Statistical analysis was by the Student's t test.
l
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Kv1.2 and Kv1.2N2 were transfected into CHO cells in thepresence of tunicamycin or into Lec1 cells. Kv1.2N2 had asimilar profile of protein expression as Kv1.2 in CHO cells withtunicamycin (Figs. 5F–H) or in Lec1 cells (data not shown). Asdiscussed previously, the upper band in Fig. 5F is a phos-phorylated protein. Furthermore, Kv1.2N2 showed a similarconductance density level as Kv1.2 when expressed in Lec1cells (Fig. 5I). These results suggest that it was an N-glycan onthe extended S3–S4 linker that was responsible for the findingand not the extended amino acid linker per se.
To address whether moving an N-glycan from the S1–S2linker to the S3–S4 linker affected steady-state activation, the
macroscopic current traces of Kv1.2 and Kv1.2N2 wererecorded by 80 ms depolarizing pulses and normalized G–Vcurveswere plotted. Kv1.2N2 showeda shorter delay to currentrise following voltage pulses and faster activation kineticscomparedwith Kv1.2 (Figs. 6A, B, and E). Furthermore, Kv1.2N2exhibited a ~16 mV hyperpolarized V1/2 shift, compared withKv1.2 (Fig. 6G), which suggests that less depolarization wasrequired to activate Kv1.2N2. The slopes of G–V curves weresimilar for both Kv1.2 and Kv1.2N2 (~−11.5 mV), whichsuggests that they have a similar voltage dependence. Tocontrol for the extended linker, Kv1.2 and Kv1.2N2 wereexpressed in glycosylation-deficient Lec1 cells. In Lec1 cells,
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Kv1.2 and Kv1.2N2 showed similar activation kinetics (Figs. 6C,D, and F), similar V1/2 values (~4.8 mV), and similar G–V curveslopes (~−16.4 mV) (Fig. 6H). This result suggests that it is theN-glycan, not the amino acids, on the extended linker that isresponsible for the effect.
To determine whether attaching an N-glycan to the S3–S4linker produced a similar effect as increasing surface negativecharge, the effects of extracellular divalent cations on V1/2
were measured by plotting G–V curves. Addition of Sr2+
(50 mM) caused a larger depolarized V1/2 shift of ~34.4 mV inthe G–V curve of Kv1.2N2 (Fig. 7C), compared with Kv1.2 wildtype, which had a smaller depolarized V1/2 shift of ~22.3 mV(Fig. 7A). Thus, it appears that anN-glycan on the extended S3–S4 linker affected theS4voltage-sensor to a greaterdegree thanan N-glycan on the S1–S2 linker (assuming the two N-glycanshave a similar structure/composition). To control for theextended linker, Kv1.2 and Kv1.2N2 were expressed in Lec1cells and the strontium screening of surface negative chargeswere performed. Similar depolarized shifts (~17mV) in theG–Vcurve were recorded for both Kv1.2 (Fig. 7B) and Kv1.2N2 (Fig.7D), and this suggests that it is the N-glycan, not the aminoacids, on the extended linker that is responsible for the effect.
These results suggest that an N-glycan on the extended S3–S4 linker of Kv1.2 would significantly alter channel activationbut it had little or no effect on channel protein expression.
2.4. A non-native N-glycan on an extended S5-P linker ofKv1.2 disrupted cell surface protein expression and function
The S5-P linker (turret) on the extracellular side of the pore isimportant for trafficking and function of Kv channels (Zhuet al., 2001; Eduljee et al., 2007). Kv1.2 does not have anative N-glycosylation site on this linker. To test whetheran N-glycan attached to this region had any effects on Kv1.2,Kv1.2N207Q was used to generate Kv1.2N3, which has anengineered N-glycosylation site in the middle of the turret(Fig. 8A). Kv1.2N3was not glycosylated (data not shown) and thelinker appears to be too short to be glycosylated. Kv1.2N4 wasthen generated, which has an extended turret with an engi-neered N-glycosylation site (Fig. 8A). Kv1.2N4 was glycosylated
Fig. 6 – Impact on functional properties when an N-glycan is atttransiently expressed in CHO cells or Lec1 cells. (A–B) Scaled whCHO cells. The cDNA amount of Kv1.2 was decreased to obtain sthen scaled so that differences in activation kinetics may be viewand 1.7 nA for Kv1.2 and Kv1.2N2, respectively. The activation kscales. Cells were held at −80 mV and depolarized to 50 mV in 10traces. (C–D) Scaledwhole cell currents for Kv1.2 (C) and Kv1.2N2 (Dwere 2.4 nA and 2.3 nA for Kv1.2 and Kv1.2N2, respectively. TheActivation kinetics of Kv1.2 constructs in CHO cells (E) and Lec1 crise from 10% to 90% of its peak current. Mean values are plotted(G–H) The normalized conductance–voltage (G–V) curves of Kv1.2are plotted as a function of voltage with error bars indicating SE,significant hyperpolarizedV1/2 shift of the G–V curve but had littleof Kv1.2N2 were −18.3±2.8 mV and −11.9±1.1 mV (n=9), comparV1/2 valueofKv1.2N2 is significantly different fromKv1.2 (p<0.05),(p>0.05). In Lec1 cells (H), the G–V curves of Kv1.2N2 and Kv1.2and −16.6±1.5 mV (n=9), respectively, compared with 5.1±0.9 aof Kv1.2N2 and Kv1.2 are not significantly different (p>0.05). T
but with only an immature N-glycan — the 63 kDa band wassensitive to both Endo H and PNGase F (Fig. 8B). Kv1.2N4 haddecreased surface protein level, total protein level, and relativesurface expression compared with Kv1.2 (Fig. 8C–E), suggestingthat the mutant had a decreased protein stability and adecreased trafficking efficiency. Furthermore, Kv1.2N4 did notexhibit detectable voltage-gated currents (data not shown).Since the N-glycan on the extended S5-P linker is an immaturetype, a direct comparison with Kv1.2, which contains a mixtureof mature and immature N-glycans, is unwarranted. Thus,further investigationsonKv1.2N4werenotperformed.However,these results suggest that the S5-P linker of Kv1.2 is a sensitiveregion, andattaching anN-glycan (evenan immature type) and/oraddingadditional aminoacids to itappears todisrupt bothcellsurface protein expression and function of Kv1.2 channel.
3. Discussion
Kv1 channels contain three extracellular linkers, denoted asthe S1–S2, S3–S4, and S5-P linkers. Only the S1–S2 linker has anative N-glycan at a highly conserved position. In this study,we investigated why an N-glycan is only attached to thisconserved position on the S1–S2 linker of Kv1 channels andwhy their S3–S4 or S5-P linkers do not contain N-glycosylationconsensus sites and possibly native N-glycans.
3.1. A native N-glycan at a conserved position on theS1–S2 linker of a Kv1 channel is required for normalfolding and trafficking to the cell surface
Kv1.1–Kv1.5 andKv1.7 channelshaveanativeN-glycanon theirS1–S2 linkers at similar positions (Zhu et al., 2003). Our resultsuggests that the fixed position for this N-glycanmay be due tothe requirement for wild type protein stability, folding, and/orprotein trafficking to the cell surface. When an N-glycan wasplaced on the N-terminal or middle sections of the accessibleregion of the S1–S2 linker, the surface protein levels of Kv1.2 orKv1.4 decreased significantly, whereas an N-glycan at thesedifferent positions affected function similarly as if it were at its
ached to the S3–S4 linker of Kv1.2. Kv1.2 constructs wereole cell currents from Kv1.2 (A) and Kv1.2N2 (B) transfectedomewhat similar size peak currents as Kv1.2N2. Traces wereed directly. Peak currents before scaling at 50 mVwere 2.0 nAinetics for Kv1.2 constructs are shown at two different timemV increments for 80 ms; or on the right side for expanded) transfected Lec1 cells. Peak currents before scaling at 50mV
right panels show the traces at an expanded time scale. (E–F)ells (F). Rise time is the time for an activating current trace toas a function of voltage with error bars indicating SE, n=8–12.constructs in CHO cells (G) and in Lec1 cells (H). Mean valuesn=8–9. In CHO cells (G), Kv1.2N2 was associated with achange in the slope comparedwith Kv1.2. TheV1/2 and slopeed with −2.6±1.0 mV and −11.2±1.0 mV (n=7) for Kv1.2. Thewhereas the slopeof it is not significantly different from Kv1.2overlapped. The V1/2 and slope of Kv1.2N2 were 4.6±2.5 mVnd −16.2±0.9 mV (n=8) for Kv1.2. The V1/2 values and slopeshe statistical analysis was by the Student's t test.
Fig. 7 – Effects of extracellular strontium (Sr2+) on theV1/2 of Kv1.2 constructs. The extracellular divalent cation Sr2+ was used totest the effect of screening negative surface charges on Kv1.2 constructs. Mean values are plotted as a function of voltage witherror bars as SE. (A–B) The normalized conductance–voltage curves for Kv1.2 in CHO cells (A) and in Lec1 cells (B). 50 mMSr2+ caused a depolarized shift in the G–V curve for Kv1.2. In CHO cells (A), V1/2 was shifted from −2.6±1.0 to 19.7±2.1 mV(n=11, p<0.05), with a net shift of ~22.3 mV. In Lec1 cells (B), V1/2 was shifted from 5.1±0.9 to 21.8±2.8 mV (n=11, p<0.05), witha net shift of ~16.7 mV. (C–D) The normalized conductance–voltage curves for Kv1.2N2 in CHO cells (C) and in Lec1 cells (D).50 mM Sr2+ caused a depolarized shift in the G–V curve of Kv1.2N2. In CHO cells (C), V/1/2 was shifted from −18.3±2.8 to 16.0±0.7 mV (n=12, p<0.05), with a net shift of ~34.3 mV. In Lec1 cells (D), V1/2 was shifted from 4.6±2.5 to 22.0±1.3 mV (n=12,p<0.05), with a net shift of ~17.4 mV. The statistical analysis was by the Student's t test.
24 B R A I N R E S E A R C H 1 2 5 1 ( 2 0 0 9 ) 1 6 – 2 9
native position. A number of ion channels have been shown torequire an N-glycan for proper protein stability, folding, andtrafficking (Khanna et al., 2001; de Souza and Simon, 2002;Watanabe et al., 2004). However, the positional effect of an N-glycan on the biochemical and electrophysiological propertiesof an ion channel has not been determined. The crystalstructure of Kv1.2 has been reported, but the S1–S2 linkersectionwasnot resolved (Long et al., 2005). OurN-glycosylationsite scanning experiments suggest that the S1–S2 linker ofKv1.2 is flexible and resides away from the outer membranesurface (Zhu et al., 2003). A number of protein secondarystructure programs also predicted the accessible region ofS1–S2 linker to be a coiled structure (Zhu et al., 2003). Wespeculate thatN-glycansatdifferentpositionsonacoil structurecould occupy a wide range of positions near the membranesurface. An N-glycan can influence the folding, stability, ortrafficking of a glycoprotein mainly by interacting directly withthe ER calnexin/calreticulin chaperone system (Ellgaard andHelenius, 2003; Williams, 2006) and/or other intracellularmolecules. Channels with an N-glycan at different orientationsmight bind to chaperones with different affinities, which couldlead to different impacts on protein stability and trafficking.
3.2. The S3–S4 linkers of Kv1 channels do not have nativeN-glycans presumably due to functional constraints
Our results showed that the N-glycan could be attached to anextended S3–S4 linker of Kv1.2, but it altered channel function.Models of voltage-gated K+ channels suggest that the S3–S4linker is flexible and tethered at the N-terminal end of the S4voltage-sensor (Fay et al., 2007). It has been found that the S3–S4 linker in Drosophila shakerK+ channels and in Domain 1 of L-type calcium channels was involved in channel activation(Nakai et al., 1994; Mathur et al., 1997). The length of the S3–S4linker is variable among different channels, ranging from 6amino acids in rat Kv3.1 to 25 amino acids in shaker K+
channel. A voltage-gated K+ channel has not been documen-ted to be N-glycosylated on this linker. However, native N-glycosylation sites were found in the S3–S4 linkers of severalK+ channels, for instance, rat Kv2.1 (Misonou et al., 2005),Drosophila shab K+ channels (Mathur et al., 1997), and murineBK channel (Hagen and Sanders, 2006); but these sites are notN-glycosylated presumably because they are too close to thepredicted S3 transmembrane domain. The S3–S4 linker ofKv1.2 is 16-amino acid long and does not contain a native N-
Fig. 8 – Impact on cell surface protein levels and functional properties when an N-glycan is attached to the S5-P linker ofKv1.2. Kv1.2 constructs were expressed in CHO cells. The surface expression levels were estimated bybiotinylation/immunoblotting and the total protein levels were estimated by immunoblotting. (A) S5-P linker sequences ofKv1.2 constructs. N-glycosylation sites are underlined. (B) Endo H and PNGase F treatments of Kv1.2N4 proteins. The numberto the left of the figure denotes the electrophoretic mobility in kDa. The mature and immature bands were definedas in Fig. 2. (C) Immunoblot analysis of surface proteins. The mean of Kv1.2N4 was normalized to Kv1.2, which was taken as100.0±SE, n=3. (D) Immunoblot analysis of total proteins. The mean of Kv1.2N4 was normalized to Kv1.2, which was takenas 100.0±SE, n=3. (E) Relative surface expression. It was estimated by dividing the surface protein (C) by the total protein (D).The surface protein, total protein, and relative surface expression levels of Kv1.2 and Kv1.2N4 are statistically different (p<0.05).The statistical analysis was by the Student's t test.
25B R A I N R E S E A R C H 1 2 5 1 ( 2 0 0 9 ) 1 6 – 2 9
glycosylation site. We suggest that the S3–S4 linker of Kvchannels does not have an N-glycosylation site because ofits close proximity to the S4 voltage-sensor and its involve-ment in channel activation. Our data showed that attachingan N-glycan to an extended S3–S4 linker induced fasteractivation, shifted the V1/2 to a negative voltage, andincreased sensitivity to extracellular Sr2+, compared withKv1.2 wild type, which has an N-glycan on its S1–S2 linker.Lec mutant experiments eliminated the possibility that theamino acids added to the middle section of the S3–S4 linkerwas the major cause of altered channel function. Similarresults were observed in Shaker K+ channels. Lengthening ofthe S3–S4 linker by addition of a short amino acid region
caused only small alterations in channel function, and theiractivation voltages and kinetics were similar to that of wildtype (Mathur et al., 1997). The majority of Kv1.2 or Kv1.2N2channels in the plasma membrane were mature proteinswith complex type N-glycans which contain significantamount of sialic acids (data not shown). Previous work hassuggested that the negative charged sialic acids influencethe effective surface potential sensed by the S4 voltage-sensor and this affected the channels V1/2 and activationkinetics (Thornhill et al., 1996;Castillo et al., 1997; Bennett,2002). An N-glycan on the S3–S4 linker may be closer to theS4 voltage-sensor compared with the S1–S2 linker, and thus,it could generate a greater negative surface potential on the
26 B R A I N R E S E A R C H 1 2 5 1 ( 2 0 0 9 ) 1 6 – 2 9
channel and change its voltage dependence and activationkinetics. Kv1–Kv4 channels have V1/2 values ranging from−35 mV to +35 mV as well as different activation kinetics(Conley and Brammar, 1999). We speculate that if Kv1–Kv4channels had native N-glycans on their S3–S4 linkers thentheir ranges of V1/2 values would be significantly shifted inthe hyperpolarized direction and their activation kineticswould be faster. Changes in these parameters would bepredicted to alter cell excitability and signaling. We suggestthat voltage-gated K+ channels lack N-glycosylation consen-sus sites on their S3–S4 linkers and/or have relatively shortlinkers due to functional restrictions.
3.3. The S5-P linkers of Kv1 channels do not have nativeN-glycans presumably due to structural and/orfunctional constraints
Native N-glycosylation consensus sites are found in the S5-Plinker (turret region) on Kv3.3, Kv4.1, and Kv10.1 (Conley andBrammar, 1999), but only Kv10.1 has a native N-glycan here.Kv10.1 has a relatively long S5-P linker which contains two N-glycosylation sites. One site has a mature N-glycan, whereasthe other site has an immature N-glycan— only themature N-glycan affected Kv10.1 trafficking and function (Napp et al.,2005). However, most Kv channels have relatively short S5-Plinkers that do not contain native N-glycosylation sites. Forinstance, all the members of Kv1 channels have a 13-aminoacid long S5-P linker without an N-glycosylation site. Our datashowed that an immature N-glycan could be attached to anextended S5-P linker of Kv1.2, but the channel exhibitedsignificantly disrupted biochemical properties and wedetected no voltage activated currents. We did not performfurther investigations on Kv1.2N4 due to its immature type N-glycan. Therefore, the effect we recorded could be due to theN-glycan and/or the extended amino acid sequence that weadded. The amino acids in the S5-P linker has been shown tomodulate slow inactivation in Kv1.5 (Eduljee et al., 2007) andinfluence trafficking in Kv1.1 and Kv1.4 (Manganas et al., 2001;Zhu et al., 2001). Thus, either attaching an N-glycan or addingamino acid sequence to this linker could disrupt channelstructure and function. These findings suggest that the S5-Plinker of Kv1 channels is a region that is involved in regulatingchannel trafficking and function, and placing an N-glycanand/or adding a amino acid sequence on this linker wouldsignificantly disrupt channel activities.
3.4. Conclusions
Our data indicate that (1) an N-glycan at a conserved positionon the S1–S2 linker of Kv1 channels is required for wild typeprotein stability and trafficking to the cell surface, probablydue to structural restrictions, (2) the S3–S4 linker of Kv1channels is not suitable for N-glycosylation, probably due tofunctional restrictions, and adding an N-glycan to this linkerwould significantly alter channel function but not trafficking,and (3) the S5-P linker of Kv1 channels is a sensitive region,and adding an N-glycan and/or a amino acid sequence to thisregion would disrupt both function and trafficking. Wesuggest that Kv1 channels have an N-glycan at a conservedposition on the S1–S2 linker to overcome the constraints for
proper folding, trafficking, and function that occur when anN-glycan is at a non-native position.
4. Experimental procedures
4.1. Tissue culture cells
Chinese hamster ovary (CHO) pro5 cells and the glycosylation-deficient CHO mutant (Lec1) cells (Stanley and Siminovitch,1977) were purchased from American Type Culture Collection(Manassas, VA). Cells were cultured in Dulbecco's modifiedEagle's medium, or minimum Eagle's medium, supplementedwith 0.35 mM proline, 2 mM glutamine, 100 IU/ml penicillin,100 μg/ml streptomycin, and 10% fetal bovine serum at 37 °Cunder 5% CO2. CHO cells do not express endogenous voltage-gated K+ channels and are a good system for exogenous K+
channel expression (Yu and Kerchner, 1998).
4.2. Molecular biology
Rat brain Kv1 complementary DNAs (cDNAs) were kindlyprovided by Dr. O. Pongs (Ruhr-Universitaet Bochum, Bochum,Germany) (Stuhmer et al., 1989). The polymerase chainreaction (PCR) was employed to engineer Kv1 cDNA to containa 5′ Kozak enhanced ribosomal-binding sequence (CCACC)before the start methionine and to eliminate endogenous 5′-or 3′-untranslated regions. Therefore, the stabilities andtranslation abilities of mRNAs transcribed from all cDNAs incell lines are predicted to be similar. Site-directedmutagenesiswas performed by replication mutagenesis, according to themanufacturer's procedure (Stratagene, La Jolla, CA). Comple-mentary pairs of mutagenic oligonucleotides were used tocreate mutations in Kv1 constructs. The cDNAs generatedfrom PCR or replication mutagenesis were subcloned into theeukaryotic expression vector, pcDNA3 (Invitrogen, Carlsbad,CA), and the integrity of constructs was confirmed by DNAsequencing at the Biotechnology Resource Center, CornellUniversity, Ithaca, New York.
CHO cells or Lec1 cells were transiently transfected with0.5 μg of Kv1 wild type or Kv1 mutant cDNA in 35-mm culturedishes using LipofectAMINE Plus as recommended by themanufacturer (Invitrogen, Carlsbad, CA). Cells were incubatedfor 20 h post-transfection before theywere processed since themaximal cell surface expression levels for Kv1.2 and Kv1.4were found at ~15 h post-transfection (data not shown).Nontransfected CHO cells or Lec1 cells showed no detectablesignals with Kv1.2 and Kv1.4 antibodies on immunoblots (datanot shown).
4.3. SDS-PAGE and immunoblotting
Proteins (~20 μg per lane) were separated on 9% sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) and transferred to polyvinylidene difluoride (PVDF)membranes (GE Healthcare Life Sciences, Piscataway, NJ) at12 V for 1 h. The PVDF membranes were blocked for 30 minwith 5% nonfat milk in phosphate-buffered saline (PBS) plus0.1% Tween 20 and incubated overnight in primary antibodies(Kv1 mouse monoclonal antibodies at 1:1000) and followed by
27B R A I N R E S E A R C H 1 2 5 1 ( 2 0 0 9 ) 1 6 – 2 9
horseradish peroxidase-linked anti-mouse secondary antibo-dies (1:15,000; 1.5 h at room temperature). Kv1 proteins weredetected on blots using enhanced chemiluminescence detec-tion kit (Amersham Biosciences, Pittsburgh, PA) and pre-flashed X-ray film (AR5, Eastman Kodak, Rochester, NY). Afilm image was captured with a Microtex 8700 scanmaker andanalyzed with National Institutes of Health Image 1.6 soft-ware. A standard curve using different dilutions of proteinsfrom transfected cells indicated that immunoblot signals werelinear over at least a 1–20-fold concentration range (Zhu et al.,2001).
4.4. Cell surface and total protein analysis
Cell surface biotinylation of intact cells has been described(Zhu et al., 2005). At 20–24 h post-transfection, cells werewashed three times with ice-cold PBS containing 0.1 mMCaCl2 and 1 mM MgCl2 and incubated with 10 mM NaIO4 inthe dark for 20 min to oxidize cell surface carbohydrates. Thecells were then washed and incubated with 2 mM hydrazide–LC–biotin (Pierce Biotechnology, Rockford, IL) in 100 mMsodium acetate (pH 5.5) for 15 min at 4 °C, followed bywashing in PBS with 10 mM Tris to remove the biotinylationreagent. The cells were collected in 1 ml of ice-cold lysisbuffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate,50 mM Tris, pH 7.4, 10 μM pepstatin A, 10 μM leupeptin, 1 mM1,10-phenanthroline, and 0.2 mM phenylmethylsulfonylfluoride) and rocked for 30 min at 4 °C. A sample wassaved for total protein immunoblotting. The lysate was thencentrifuged and the resulting supernatant was incubatedwith 50 μl of streptavidin–agarose beads (1:1 slurry; PierceBiotechnology, Rockford, IL). After rocking the reactionmixtures gently overnight at 4 °C, biotin–streptavidin–agar-ose complexes were harvested by centrifugation. The beadswere washed, eluted with Laemmli sample buffer, and runon 9% SDS-PAGE for immunoblotting with Kv1.2 or Kv1.4antibodies (Upstate Biotechnology, Lake Placid, NY). ThePVDF membranes containing surface proteins were strippedand re-blotted with glucose-transporter1 antibodies (Abcam,Cambridge, MA) to control for surface protein biotinylationefficiencies (data not shown). The PVDF membranes contain-ing total proteins were stripped and re-blotted with actinantibodies (clone AC-40, Sigma, Saint Louis, MO) to control forprotein loading amount (data not shown). Transfectionefficiencies were routinely checked by cotransfecting with0.1 μg of green fluorescence protein (GFP) cDNA and perform-ing immunoblots with anti-GFP antibodies (Clontech, PaloAlto, CA).
4.5. Tunicamycin treatment
Tunicamycin (Sigma-Aldrich, St. Louis, MO), an antibiotic thatinhibits N-glycosylation, was dissolved in dimethyl sulfoxide(DMSO) and diluted in prewarmed culture medium to a finalconcentration of 2 μg/ml and added for 15 h to CHO cells 6 hpost-transfection. Control experiments were performed withculture medium containing similar amount of DMSO. Theproteins were analyzed by immunoblotting with Kv1 anti-bodies. Tunicamycin was used as a control for amino acidreplacements on Kv1 constructs. A wild type Kv1, with its
native N-glycosylation site, and Kv1 constructs, with an addedN-glycosylation site, will not be glycosylated in the presence oftunicamycin. Thus, in the presence of tunicamycin it ispredicted that all engineered Kv1 constructs with N-glycosyla-tion sites will have similar surface protein and total proteinlevels as wild type, if the amino acid replacement per se didnot affect these parameters.
4.6. Glycosidase treatment
The peptide-N-glycosidase F (PNGase F) and endoglycosidaseH (Endo H) were used as recommended by the manufacturer(Boehringer Mannheim Biochemicals, Indianapolis, IN). ForPNGase F digestion, 10 μl of cell lysatewas incubated overnightat 37 °Cwith 3 U of PNGase F in 5 μl of buffer (20mMphosphatebuffer, pH 7.0, 10 mM ethylene diamine tetra-acetic acid, and0.05% Triton X-100). For Endo H, 10 μl of cell lysate wasincubated 30 min at 37 °C with 0.3 U of Endo H in 5 μl of buffer(25 mM sodium citrate buffer, pH 5.7, and 0.05% Triton X-100).Both reactions were stopped by the addition of Laemmlisample buffer and the products were analyzed by electro-phoresis on 9% SDS-PAGE.
4.7. Patch-clamping
Patch-clamp recording and analysis methods have beenextensively described in our previous papers (Watanabeet al., 2003, 2007). K+ currents were recorded from CHO andLec1 cells transiently coexpressing Kv1.2 channels withGFP, for viewing with a fluorescence microscope. Patch-clamping experiments (Hamill et al., 1981) were performedat room temperature using an Axopatch 200B amplifier(Axon Instruments, Foster City, CA) 20–30 h after transfec-tion. Patch pipettes were pulled from 8161 corning glass(Warner Instruments, Hamden, CT) to give a tip resistanceof 1.5–2.5 mΩ when filled with an intracellular solutioncontaining 70 mM KCl, 65 mM KF, 5 mM NaCl, 1 mMMgCl2, 10 mM ethylene glycol tetra-acetic acid, 5mMglucose,and 10mMN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonicacid (HEPES) (pH 7.3). The external solution contained 150 mMNaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and10 mM HEPES (pH 7.3). Whole cell currents were activated byincremental depolarization to 50mV fromaholding potential of−80 mV. Nontransfected CHO or Lec1 cells showed either no orat most 100 pA of endogenous K+ current at maximumactivating voltages of 50 mV. Leak and capacitance currentswere subtracted by standard P/n procedure. Series resistancewas compensated to 85%. Maximum peak conductance values(G) were obtained from the mean value of the peak current (I)using Ohm's law (G= I/(Vp−EK)).Vp denotes the applied voltage,and EK denotes Nernst K+ equilibrium potential, which ispredicted to be −83 mV.
4.8. Statistical evaluation
Statistical analysis was performed by Student's t test or one-way analysis of variance (ANOVA) and Tukey's multiplecomparison tests. A P value<0.05 was considered significantin a statistical test. Data are shown as means±SE (standarderror of the mean).
28 B R A I N R E S E A R C H 1 2 5 1 ( 2 0 0 9 ) 1 6 – 2 9
Acknowledgments
This research was supported by National Institutes of HealthGrant USA NS048906 (W.B.T.) and the American HeartAssociation with a Grant-in-Aid. (W.B.T).
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