Characterization of N-glycosylation consensus sequences in the Kv3.1 channel Natasha L. Brooks, Melissa J. Corey and Ruth A. Schwalbe Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC, USA Voltage-gated K + channel (Kv3.1) plays a fundamen- tal role in neuronal excitability and lymphocyte differ- entiation [1–6], and belongs to the Kv3 subfamily of the voltage-gated K + channel (Kv) supergene family [7]. Upon stimulation, the voltage-dependent gate opens and potassium ions flow out of the cell, indu- cing negative intracellular voltage, and termination of excitation [8]. Based on hydropathy plots, Kv3.1 has six transmembrane segments (S1–S6) and cytoplasmic N- and C-termini (Fig. 1A). The segments between S1–S2 and S3–S4 are extracytoplasmic loops, and those between S2–S3 and S4–S5 are cytoplasmic loops. Each of the Kv3.0 family members and their splice variants contain two conserved, native N-glycosylation sites in the S1–S2 linker. Rat and human Kv3.1 pro- teins have two native N-glycosylation consensus sequences running from amino acid residues 220 to Keywords brain; glycosylation; K + channel; topology, trafficking Correspondence R. A. Schwalbe, Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA Fax: +1 252 744 3383 Tel: +1 252 744 2034 E-mail: schwalber@mail.ecu.edu (Received 25 August 2005, revised 18 May 2006, accepted 23 May 2006) doi:10.1111/j.1742-4658.2006.05339.x N-Glycosylation is a cotranslational and post-translational process of pro- teins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage- gated K + channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, par- tially purified proteins were digested with either PNGase F or endoglycosi- dase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q ⁄ N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q ⁄ N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q ⁄ N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q ⁄ N229Q. These results demonstrate that the S1–S2 linker is topologically extracellular, and that N-glycosylation influen- ces the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the func- tional Kv3.1 at the cell surface. Abbreviations CDGS, carbohydrate-deficient glycoprotein syndromes; Endo H, endoglycosidase H; ER, endoplasmic reticulum; G–V plot, conductance– voltage plot; Kv, voltage-gated K + channel; KvAP, voltage-gated K + channel of Aeropyrum pernix; PM, plasma membrane; PNGase F, peptide N glycosidase F; Sf9, Spodoptera frugiperda; TM, tunicamycin. FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 3287 222 (NKT) and from 229 to 231 (NGT) in the S1–S2 linker, and they share 100% sequence identity in this region (Fig. 1B). A recent X-ray structure of a Kv from Aeropyrum pernix (KvAP) suggested that the S1–S2 linker resides in the membrane for all Kvs [9]. Comparison between the S1–S2 linker of KvAP and mammalian Kvs may be difficult because of differences in the length of their S1–S2 linkers and the N-glycosy- lation consensus sequences within this segment for Kv3.0s and Kv1.0s (Fig. 1C). N-Glycosylation is a cotranslational and post-trans- lational modification found on extracellular segments of membrane proteins and is important for protein maturation, trafficking, and function [10–13]. The N- glycosylation consensus sequence is AsnXxxSer ⁄ Thr, where the central residue cannot be a Pro residue. Membrane protein segments are only glycosylated when they are translocated to the luminal side of the endoplasmic reticulum (ER) membrane, and therefore occupancy of an N-glycosylation site designates a region of extracellular topology [11,12]. Defects in the attachment of oligosaccharides to protein give rise to mental and psychomotor retardation, dimorphisms, and blood coagulation defects [14,15]. Carbohydrate- deficient glycoprotein syndromes (CDGS) I–IV are a group of disorders characterized by the presence of abnormal oligosaccharides on many glycoproteins [16,17]. The occurrence of CDGS emphasizes that proper glycosylation of both membrane and secretory glycoproteins are essential for normal development and health [18,19]. More recently, it has been sugges- ted that ER stress is linked to several human neuron- al diseases [20], and therefore it may be that abnormal glycosylation processing of proteins contri- butes to these diseased states as well. Here we have examined whether the native N-gly- cosylation sites are utilized in rat brain and infected Sf9 cells, and the role that occupancy of these sites has in the expression of functional Kv3.1s at the cell surface of Sf9 cells. Immunoband patterns of wild- type Kv3.1, N220Q ⁄ N229Q, N229Q, and N220Q, in the absence and presence of tunicamycin (TM), endo- glycosidase H (Endo H), or peptide N glycosidase F (PNGase F), revealed that both sites in Kv3.1 were occupied by N-linked oligosaccharides. Patch clamp measurements and cell fractionation showed that the unglycosylated Kv3.1, N220Q ⁄ N229Q, is targeted to the plasma membrane, like wild-type Kv3.1. However, whole cell currents of N220Q ⁄ N229Q revealed slower activation kinetics and a small positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of activation for the partially glycosylated Kv3.1s, N229Q and N220Q, appeared similar to that of N220Q ⁄ N229Q. Our findings demonstrate that the S1–S2 linker of Kv3.1 is in an extracellular aqueous environment. Additionally, they demonstrate that N-glycosylation influences the open- ing of the voltage-dependent gate of Kv3.1, suggest- ing that vacant sites alter the folding and maturation of Kv3.1 at the cell surface. N C 220 229 S1 S2 S3 S6 S5 S4 A Rat 210 ETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTY Human 210 ETHERFNPIV NKTEIENVRNGTQVRYYREAETEAFLTY B Kv3.1 P25122 210 ETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTY Kv1.1 P10499 187 ETLPELKDDKDFTGTIHRIDNTT VIYTSNIFTDP Kv1.2 P63142 183 ETLPIFRDENEDMHGGGVTFHTYSNST IGYQQSTSFTDP Kv1.4 P15385 329 ETLPEFRDDRDLIMALSAGGHSRLLNDT SAPHLENSGHTIFNDP Kv1.5 P19024 261 ETLPEFRDERELLRHPPVPPQPPAPAPGINGS VSGALSSGPTVAPLLPRTLADPF KvAP Q9YDF8 64 SGEY C Fig. 1. Topological model of Kv3.1 and amino acid sequences of the S1–S2 linker of Kvs. (A) Topology of a Kv3.1 monomeric unit. Black circles represent the Asn of native N-glycosylation consensus sites N220 and N229. Branched structures represent the attachment of oligosaccharide at native sites. (B) Sequence identity between Kv3.1 S1–S2 linkers from rat (P25122) and human (P48547). (C) Comparison of the S1–S2 amino acid sequence of eukaryotic Kvs and prokaryotic KvAPs. The Kv name corres- ponding to the adjacent S1–S2 amino acid sequence is indicated in bold and is fol- lowed by the accession number. Conserved, native N-glycosylation sites are shown as underlined font. The italicized number indi- cates the first residue of the S1–S2 linker. Characterization of glycosylation sites in Kv3.1 N. L. Brooks et al. 3288 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS Results Occupancy of the two native N-glycosylation sites Rat brain membranes were digested with PNGase F, and then analyzed by western blotting. PNGase F is an enzyme that removes a wide range of N-linked oligosaccharides from proteins [21]. Native Kv3.1 migrates as a diffuse immunoband (about 109 kDa) which is much larger than its calculated molecular mass of 66 kDa (Fig. 2). This migration pattern of native Kv3.1 suggests that the protein undergoes a cotranslational or post-translational modification. To show that the modification was indeed a result of attachment of N-linked oligosaccharides, rat brain membranes were incubated with PNGase F. The Kv3.1 immunoband (about 81 kDa) migrated much faster, indicating that Kv3.1 undergoes N-glycosylation in rat brain membranes. To further verify specificity of the Kv3.1b antibody, membranes isolated from Sf9 cells infected with recombinant baculovirus that enco- ded the Kv3.1b cDNA were immunoblotted (Fig. 2). The electrophoretic migration pattern of wild-type Kv3.1 revealed a predominant immunoband at about 87 kDa and two lower faint bands. The lowest band migrated to about 77 kDa, and the middle band was at about 81 kDa. Only the two lowest bands were detected when Sf9 cell membranes were treated with PNGase F, suggesting that the top two bands are gly- cosylated protein. Taken together, these results demon- strate that Kv3.1 is N-glycosylated in rat and insect cells, and that the type of N-linked oligosaccharide differs. To directly demonstrate that both of the absolutely conserved N-glycosylation consensus sequences were utilized, they were removed independently (N229Q and N220Q) and simultaneously (N220Q ⁄ N229Q) by conserved substitutions of the Asn residues with Gln residues. In addition, an M2 FLAGÒ epitope was attached to the C-terminus and was utilized for purifi- cation and identification of the various Kv3.1 proteins. Wild-type Kv3.1, N229Q, N220Q and N220Q ⁄ N229Q were M2 immunoaffinity purified from whole cell lysates of Sf9 cells infected in the absence and presence of TM, and then immunoblotted using anti- ++ _ _ Anti-Kv3.1b rat brain membranes Sf9 cell membranes PNGase F: Fig. 2. N-Glycosylation of Kv3.1 in rat brain tissue and Sf9 cell membranes. Rat brain membranes and partially purified Sf9 pro- teins were untreated (–) or treated (+) with PNGase F, resolved by SDS ⁄ PAGE and immunoblotted, as indicated. The arrows of each panel indicate migration of glycosylated (upper arrow) or unglycosyl- ated (lower arrow) Kv3.1 protein. Ovals represent Kaleidoscope TM protein standards (top to bottom in kDa): 250, 150, 100, and 75. Endo H: ____ ++++ TM : Wt N220Q/ N229Q N229Q N220Q Kv3.1: ____ ++++ Anti-FLAG Wt N229Q N220Q N220Q/ N229Q + +++ _ _ _ _ Kv3.1: TM: A B Anti-Kv3.1b Fig. 3. Detection of high-mannose oligosaccharides in Sf9 cell membranes. Sf9 cells were infected with recombinant baculovirus containing wild-type Kv3.1, N229Q, N220Q or N220Q ⁄ N229Q in either the absence (–) or the presence (+) of 25 lgÆmL )1 tunicamy- cin (TM). Proteins were transferred and probed with anti-Kv3.1b (A) or anti-FLAG (B, upper panel). Partially purified Kv3.1 protein was treated (+) with endoglycosidase H (Endo H) (B, lower panel). The arrows in each panel indicate migration of fully glycosylated (upper), partially glycosylated (middle) or unglycosylated (lower) Kv3.1 protein. In all cases, proteins were partially purified from whole cell lysates using M2-agarose. Ovals represent molecular mass stand- ards (top to bottom in kDa): 250, 150, 100, and 75. N. L. Brooks et al. Characterization of glycosylation sites in Kv3.1 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 3289 Kv3.1b (Fig. 3A) and M2 anti-FLAG (Fig. 3B). TM inhibits the oligosaccharyltransferase that carries out the initial step of the N-glycosylation pathway in the ER lumen [22]. As mentioned above, wild-type Kv3.1 migrates as three immunobands, with the upper band as the predominant band. When Sf9 cells expressing wild-type Kv3.1 were treated with TM, only the lowest immunoband was observed. The single Kv3.1 mutants, N229Q and N220Q, migrated as doublets which appear to correspond to the lower two immunobands of wild-type Kv3.1. In both cases, the upper band was darker than the lower band. Additionally, the upper band was not visible in the presence of TM. A single immunoband was detected for N220Q ⁄ N229Q, which migrated to a similar position as the lowest faint band observed for wild-type Kv3.1 and the lower faint band of the single mutants, and furthermore, the immuno- band did not shift in the presence of TM. To verify that wild-type Kv3.1 was modified by a high-mannose oligosaccharide typical of Sf9 cells, not a complex oligosaccharide [23], N-linked oligosaccharide was removed by Endo H treatment of partially purified Kv3.1 protein (Fig. 3B). When partially purified wild- type Kv3.1, N229Q and N220Q proteins were digested with Endo H, the lowest band becomes the predomin- ant form in all three instances. The band observed for N220Q ⁄ N229Q does not shift in the presence of Endo H. These results indicate that the upper band of wild-type Kv3.1 represents the situation when both glycosylation sites are occupied by high-mannose-type oligosaccharides, the middle band represents one occu- pied site, and the lowest band is the unglycosylated monomer. Glycosylated and unglycosylated forms of Kv3.1 are targeted to the plasma membrane Infected Sf9 cells expressing wild-type Kv3.1 and N220Q ⁄ N229Q were fractionated into three distinct fractions [24,25]. Subsequently, Kv3.1 protein was M2 immunoaffinity purified from each fraction (Fig. 4A). A predominant immunoband was detected for wild- type Kv3.1 in all three distinct fractions. Two faint lower bands were clearly observed in the ER fraction, while in the other two fractions only the lower faint band was observed. The totally unglycosylated form of Kv3.1, generated by mutating Asn residues at positions 220 and 229 to Gln residues (N220Q ⁄ N229Q, Fig. 4A) or by treating Sf9 cells expressing wild-type Kv3.1 with TM (Fig. 4B), was also observed in the plasma mem- brane. These results indicate that N-glycosylation is not required to transport Kv3.1 to the plasma mem- brane. Functional unglycosylated Kv3.1 is at the cell surface Whole cell currents of infected Sf9 cells expressing either wild-type Kv3.1 or N220Q ⁄ N229Q were observed when the membrane potential was depolar- ized beyond ) 10 mV and current amplitudes reached saturation at membrane potentials beyond +40 mV (Fig. 5A,B, top panel). The patterns of these inactivat- ing, voltage-dependent K + currents were typical of a delayed rectifier, and were similar to those expressed by wild-type Kv3.1 in Xenopus oocytes [1,3,4,26–28] and other heterologous expression systems [29–31]. To show that channel densities at the cell surface for wild- type Kv3.1 (I max ⁄ cap is 140 ± 29 pA ⁄ pF, n ¼ 13) and N220Q ⁄ N229Q (I max ⁄ cap is 156 ± 36 pA ⁄ pF, n ¼ 11) were comparable, the maximum current amplitude was determined and divided by the cell capacitance. Differ- ences between the two forms could be identified when the voltage dependence for channel activation was analyzed. The membrane conductance vs. applied test potential indicated that more depolarization was required for 50% of the N220Q ⁄ N229Q channels (V 0.5 [test potential at which g/g max ¼ 0.5] is 20.5 ± 0.6 mV, n ¼ 11) to reach activation than for wild-type Kv3.1: A Fraction: PM Golgi ER PM Golgi ER Wt N220Q/N229Q B Fraction: PM Golgi ER Wt Kv3.1 +TM Kv3.1: Fig. 4. Glycosylated and unglycosylated Kv3.1 proteins are deliv- ered to the plasma membrane. (A) Plasma membrane (PM), Golgi apparatus (Golgi) and endoplasmic reticulum (ER) fractions from Sf9 cells infected with the indicated Kv3.1 baculovirus were isola- ted by sucrose density gradients. Protein was M2-affinity agarose purified from each fraction and immunoblotted. (B) M2-agarose affinity purified Kv3.1 protein from membrane fractions of Sf9 cells expressing wild-type Kv3.1 in the presence of 25 lgÆmL )1 tunica- mycin (TM). The top two arrows denote where glycoforms would be and the bottom arrow represents the aglycoform. Characterization of glycosylation sites in Kv3.1 N. L. Brooks et al. 3290 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS Kv3.1s (V 0.5 is 16.6 ± 0.7 mV, n ¼ 13). Additionally, slightly fewer channels were activated as the applied voltage was increased for unglycosylated Kv3.1 (slope of normalized current voltage relationship, dV,is 9.3 ± 0.4 mV for N220Q ⁄ N229Q, n ¼ 11) than for glycosylated Kv3.1 (dV is 8.1 ± 0.4 mV for wild-type Kv3.1, n ¼ 13). A range of values for Vm 0.5 from 10 mV to 18 mV, and for dV from 8 mV to 11 mV, of wild-type Kv3.1 have previously been reported in various heterologous expression systems [30]. The activation kinetics of wild-type Kv3.1 expressed in vitro and in vivo is quite rapid [30,31]. When the whole cell current tracings were normalized at each potential from + 20 mV to + 50 mV for wild-type Kv3.1 and N220Q ⁄ N229Q, and then placed on top of each other, it was observed that the activation kinetics were somewhat slower for N220Q ⁄ N229Q than for wild-type Kv3.1 (Fig. 6A). AB 25 ms 25 ms 0.5 nA 0.5 nA g/gmax Voltage (mV) C -40 -20 0 20 40 60 80 100 0.0 0.2 0.4 0.6 0.8 1.0 Wt Kv3.1 N220Q/N229Q Fig. 5. Functional expression of wild-type Kv3.1 and the N220Q ⁄ N229Q mutant. Whole cell currents were produced by depolarizing voltage pulses from a holding potential of ) 50 mV to levels ranging from ) 40 to +100 mV in 10 mV increments. Repre- sentative tracings are shown from Sf9 cells infected with (A) wild- type Kv3.1 and (B) N220Q ⁄ N229Q. (C) Corresponding Boltzmann plots. Wild-type Kv3.1 (V 0.5 ¼ 16.6 ± 0.7, dV ¼ 8.1 ± 0.4, n ¼ 13) data are represented by (d) and N220Q ⁄ N229Q (V 0.5 ¼ 20.56 ± 0.6, dV ¼ 9.3 ± 0.4, n ¼ 11) data are represented by n. The Boltzmann isotherm G ¼ G max ⁄ [1 + exp(V 0.5 ) V) ⁄ q] was used to fit the data, which represent ± SEM. A 25 ms tnerruc B 20 40 60 80 100 0 10 20 30 40 50 60 Voltage (mV) Rise times (ms) 20 40 60 80 100 0 10 20 30 40 Volta g e (mV) Activation time constants (ms) C Fig. 6. Comparison of activation rates in wild-type Kv3.1 and N220Q ⁄ N229Q. (A) Whole cell currents from Sf9 cells expressing wild-type Kv3.1 (solid line) and N220Q ⁄ N229Q (dashed line) were normalized at 20 mV (red), 30 mV (blue), 40 mV (purple) and 50 mV (black), and the resulting normalized currents were placed on top of each other. (B) Rise times and (C) activation time constants are shown for wild-type Kv3.1 (d) and N220Q ⁄ N229Q (n). Rise times represent the time required for the current to rise from 10% to 90% of its peak current at the indicated applied voltage. Activation time constants were determined by fitting the current traces at each potential to a single exponential. Data represent SEM. N. L. Brooks et al. Characterization of glycosylation sites in Kv3.1 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 3291 In both cases, the activation time decreased as the applied potential increased, which indicates the volt- age dependence of channel activation. The time for the current to rise from 10% to 90% of its maximum value was less for N220Q ⁄ N229Q than for wild-type Kv3.1 at the various applied potentials (Fig. 6B). Time constants for activation at similar potentials were also determined by fitting each current with a single exponential (Fig. 6C). Again, it was demonstra- ted that the activation rate for N220Q ⁄ N229Q is slower than that for wild-type Kv3.1. The deactiva- tion kinetics of wild-type Kv3.1 (time constant deacti- vation, s off is 2.4 ± 0.9 at ) 40 mV, n ¼ 3) and N220Q ⁄ N229Q (s off is 3.6 ± 0.3 at ) 40 mV, n ¼ 3) were rapid, and similar to those previously reported in heterologous expression systems and neurons [31]. These results indicate that differences in the voltage dependence of channel activation can be measured between the glycosylated Kv3.1 (wild-type Kv3.1) and its unglycosylated counterpart (N220Q ⁄ N229Q). Previously, it has been reported that whole cell current recordings of wild-type Kv3.1 in mammalian expression systems display little saturation in current amplitude in response to large depolarization steps [29,32,33]. This noninactivating type of behavior was also observed for both glycosylated and unglycosylat- ed Kv3.1s expressed in Sf9 cells (Fig. 7A,B) but occurred less often than the inactivating currents. In the case of the noninactivating behavior, the channel densities for wild-type Kv3.1 (I max ⁄ cap is 368 ± 29 pA ⁄ pF, n ¼ 11) and N220Q ⁄ N229Q (I max ⁄ cap is 314 ± 50 pA ⁄ pF, n ¼ 9) were quite similar. How- ever, both forms of Kv3.1 had higher channel densi- ties than those that had inactivating behavior. Like the cells that expressed the inactivating type of behavior for wild-type Kv3.1 and N220Q ⁄ N229Q, the rise times at the various potentials were slower for the unglycosylated Kv3.1 than for glycosylated Kv3.1 (Fig. 7C). Moreover, the rise times were faster in those cells that expressed the noninactivating type of behavior than in those that expressed the inacti- vating type of behavior for either wild-type Kv3.1 or N220Q ⁄ N229Q. Single-channel recordings of wild-type Kv3.1 and N220Q ⁄ N229Q have long openings, and long and brief closures (Fig. 8A,B). Current amplitudes and unitary conductances of wild-type Kv3.1 and N220Q ⁄ N229Q were virtually identical (Fig. 8C), and quite similar to those in previous reports of wild-type Kv3.1 in Xenopus oocytes [26,28] and mammalian cells [29,32]. These results indicate that the current amplitudes and single-channel conductances are similar for glycosylat- ed and unglycosylated Kv3.1s. Partially glycosylated Kv3.1 mutants at the cell surface are functional The single N-glycosylation Kv3.1 mutants (N229Q and N220Q) expressed whole cell currents at applied poten- tials of ) 10 mV, and current amplitudes increased as the applied potential increased until currents reached saturation at membrane potentials beyond + 40 mV (Fig. 9A,B). The Boltzmann equation indicates that a little more depolarization is required to activate 50% of the partially glycosylated Kv3.1s (V 0.5 is 18.7 ± 1.2 mV and 20.9 ± 1.3 mV for N229Q and N220Q, respectively, n ¼ 5) than for the fully glycosyl- ated Kv3.1 (wild-type Kv3.1; Fig. 9C). The conduct- ance–voltage (G–V) slopes for N229Q (dV is 8.7 ± 0.6 mV, n ¼ 5) and N220Q (dV is 9.0 ± 0.7 mV, n ¼ 5) appear to be more similar to those for N220Q ⁄ N229Q than wild-type Kv3.1, but they were not statistically different. As for the N220Q ⁄ N229Q channel (rise times at + 20 mV, 50.5 ± 3.3 ms, and + 40 mV, 29.6 ± 2.6 ms, n ¼ 10; and activation time constants at + 20 mV, 27.7 ± 3.1 ms, and + 40 mV, 16.0 ± 20 40 60 80 100 0 10 20 30 40 Voltage (mV) Rise times (ms) C AB 1 nA 1 nA 25 ms 25 ms Wt Kv3.1 N220Q/N229Q Fig. 7. Whole cell analysis of noninactivating currents from wild- type Kv3.1 and N220Q ⁄ N229Q. Representative tracings are shown from Sf9 cells infected with (A) wild-type Kv3.1 and (B) N220Q ⁄ N229Q. (C) Rise times are shown for wild-type Kv3.1 (n ¼ 11, d) and N220Q ⁄ N229Q (n ¼ 8, n). Data represent ± SEM. Characterization of glycosylation sites in Kv3.1 N. L. Brooks et al. 3292 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 1.2 ms, n ¼ 11), the activation kinetics of N229Q (rise times at +20 mV, 54.9 ± 6.4 ms, and +40 mV, 26.3 ± 2.3 ms; and activation time constants at +20 mV, 29.6 ± 5.6 ms, and +40 mV, 12.8 ± 1.0 ms, n ¼ 5) and N220Q (rise times at +20 mV, 54.5 ± 5.2 ms; and +40 mV, 34.4 ± 2.8 ms; and acti- vation time constants at +20 mV, 30.8 ± 3.8 ms, and +40 mV, 16.7 ± 0.9 ms, n ¼ 5) were slower than those of wild-type Kv3.1 (rise times at +20 mV, 41.0 ± 2.7 ms and +40 mV, 19.4 ± 1.1 ms; and acti- vation time constants at +20 mV, 20.4 ± 1.6 ms, and +40 mV, 10.5 ± 0.8 ms, n ¼ 13). These results indi- cate that the absence of one N-linked high-mannose- type oligosaccharide can produce small changes in the voltage dependence of channel activation. Discussion The findings reported here demonstrate that both of the conserved N-glycosylation consensus sequences, in the S1–S2 linker, of Kv3.1 can be occupied by various types of oligosaccharide. Three distinct immunobands were identified for wild-type Kv3.1 expressed in Sf9 cells. The upper band was the predominant band, and the two lower bands were of minor intensity (Fig. 3). In the presence of PNGase F, Endo H, or TM, the upper band was no longer observed. When digestion was complete or cells expressing wild-type Kv3.1 were treated with TM, only the lowest faint immunoband was observed (Fig. 3). In addition, when the conserved N-glycosylation sites were removed either independ- ently or together, the immunobands corresponded to the two faint bands of wild-type Kv3.1 or the lowest faint band, respectively. These results indicate that the most prominent band of wild-type Kv3.1 expressed in insect cells represents occupancy of both glycosylation sites by simple oligosaccharides, the upper faint band represents occupancy of one site by a simple oligosac- charide, and the lowest faint band represents vacancy of both sites. A +60 mV +80 mV +100 mV 100 ms 1 pA Wt Kv3.1 N220Q/N229Q B O C 30 60 90 120 150 1 2 3 4 C Volta g e (mV) Current (pA) Fig. 8. Single-channel analysis of wild-type Kv3.1 and N220Q ⁄ N229Q. Representative single-channel recordings from infected Sf9 cells expressing (A) wild-type Kv3.1 ( d ) and (B) N220Q ⁄ N229Q (n) at various test potentials as indicated. The open state of the channel is indica- ted by O and the closed state is represented by C. (C) Current–voltage relationship of wild-type Kv3.1 and N220Q ⁄ N229Q. Single-channel conductance was 27 pS (n ¼ 8) for wild-type Kv3.1 and 24 pS (n ¼ 6) for N220Q ⁄ N229Q. Open circles denote wild-type Kv3.1 and closed circles represent N220Q ⁄ N229Q. Linear regression fit of the data was performed with a dashed line for wild-type Kv3.1 and a solid line for N220Q ⁄ N229Q. Data represent ± SEM. N. L. Brooks et al. Characterization of glycosylation sites in Kv3.1 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 3293 On the basis of conservation of initial steps in the N-glycosylation pathway and divergence of this path- way following synthesis of the common N-glycan inter- mediate, GlcNAc 2 Man 3 GlcNAc 2 -N-Asn, in insects and mammals [34], we would expect Kv3.1 to be glycosyl- ated in native tissue. A diffuse immunoband was observed in rat brain membranes that migrated much slower than would be expected from its calculated molecular mass, or that detected in Sf9 cells (Fig. 2). Previous studies are in agreement that Kv3.1 in rat brain migrates much slower than would be expected from its predicted molecular mass [35], and further- more, its migration appears to differ in various regions of the brain [36,37]. This slow migration pattern of Kv3.1 was not shown to be due to N-glycosylation. Our results show that digestion of rat brain mem- branes with PNGase F produces a much faster-migra- ting band that moves to a similar position as unglycosylated Kv3.1 expressed in Sf9 cells. Taken together, we conclude that Kv3.1 isolated from rat brain is N-glycosylated (Fig. 2), and the oligosaccha- rides are of either hybrid or complex type in composi- tion. Additionally, it may be that the composition of N-linked oligosaccharides is different in various regions of the brain. Many glycosylation studies have indicated that in order for the oligosaccharyltransferase to have access to an N-glycosylation consensus sequence of a mem- brane protein, the segment containing the tripeptide sequence must enter the lumen of the ER, which becomes the extracellular segment of the protein once it is transferred to the plasma membrane [11,38,39]. Additionally, if the site is within an extracytoplasmic loop, the segment must be larger than 30 residues, and this site must be at least 11 residues away from the membrane. Utilization of a site is also greater at an earlier time point during protein synthesis. In conjunc- tion, glycosylation of sites at positions 220 and 229 of Kv3.1 confirms the extracellular placement of the S1– S2 linker identified by hydropathy plots (Fig. 1A). This finding is also in agreement with utilization of the native glycosylation site in the S1–S2 linker of other Kvs (Fig. 1C). For example, the native glycosylation site in the S1–S2 linker of Shaker H4 [40], Kv1.1 [41], Kv1.2 [42], Kv1.4 [43] and Kv1.5 [44] were shown to be occupied by N-linked oligosaccharides. Addition- ally, utilization of introduced glycosylation sites throughout the S1–S2 segment of Kv1.2 suggested that the majority of this segment resides in the extracellular aqueous environment and that its conformation is flex- ible [42]. The Kv3.1 results demonstrated that both glycosylation sites in the S1–S2 linker were utilized, indicating that both of these regions can accommodate a conformation accessible to the oligosaccharyltransf- erase. Moreover, the large hydrophilic oligosaccharides attached to Asn220 and Asn229 would place the entire region of the S1–S2 linker outside the lipid bilayer. The structural model of bacterial KvAP places this segment in a lipid environment, and suggests that this was the case for all Kvs [9]. It is possible that the S1– S2 linker may reside in the membrane of a bacterial Kv, which would not undergo N-glycosylation, but it is unlikely that this orientation applies to all eukaryot- ic Kvs. The S1–S2 linker of KvAP is very short (four residues) compared to the longer linkers of Kv3.1 (38 residues), Kv1.1 (34 residues), Kv1.2 (39 residues), Kv1.4 (44 residues) and Kv1.5 (55 residues) (Fig. 1C). Therefore, this region in the bacterial channel is not very comparable to that of eukaryotes. The aforemen- tioned glycosylation reports, along with our report, provide strong evidence that the conserved S1–S2 linker of Kv1.1, Kv1.2, Kv1.4 and Kv1.5, along with that of Kv3.1, is topologically extracellular. The reports also suggest that the orientations of the S1 and C -40 -20 0 20 40 60 80 100 0.0 0.2 0.4 0.6 0.8 1.0 g/gmax Voltage (mV) 25 ms 0.5 nA A 0.5 nA 25 ms B N220Q N229Q Fig. 9. Functional expression of N-glycosylation single mutants. Whole cell currents are shown for Sf9 cells infected with (A) N229Q and (B) N220Q. (C) Corresponding Boltzmann plots for N229Q (. solid line; V 0.5 ¼ 18.7 ± 1.2, d V ¼ 8.7 ± 0.6, n ¼ 5) and N220Q (m dashed line; V 0.5 ¼ 20.9 ± 1.3, dV ¼ 9.0 ± 0.7, n ¼ 5) are shown here. Characterization of glycosylation sites in Kv3.1 N. L. Brooks et al. 3294 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS S2 segments are similar in all eukaryotic Kv1.0 and Kv3.0 subfamilies, but not necessarily the same between prokaryotic and eukaryotic domains. N-Glycosylation, a cotranslational and post-transla- tional process, of proteins may influence protein fold- ing, maturation, stability, trafficking, and consequently cell surface expression of functional channels [10–12]. Cell fractionation results of unglycosylated Kv3.1 produced by elimination of the two native sites, N220Q ⁄ N229Q mutant, or by treating cells expressing wild-type Kv3.1 with TM, demonstrated that targeting to the plasma membrane was not abolished. In addi- tion, whole cell current measurements of unglycosylat- ed Kv3.1 (N220Q ⁄ N229Q) and partially glycosylated Kv3.1 (N229Q and N220Q) show that they form func- tional channels at the cell surface. These results indi- cate that N-glycosylation is not required for transport of Kv3.1 to the cell surface, and thus suggest that the protein can fold and assemble into a stable functional homomultimer. However, the small changes in the acti- vation kinetics suggest that when the sites are vacant, Kv3.1 is folded slightly different in its mature structure compared to when the sites are occupied. Previous studies of Kv1.4 have also demonstrated that glycosy- lation of the native site in the S1–S2 linker was required for proper trafficking and stability [43]. On the other hand, glycosylation of the native site in the S1–S2 linker of Kv1.0s, including Shaker [40] and its mammalian homologue, Kv1.1 [41], did not affect cell surface expression. In this article, we demonstrate that the voltage dependence of Kv3.1 activation is altered by the absence of N-linked oligosaccharides. More depolar- ization (about 6 mV) was required to activate 80% of the unglycosylated Kv3.1s (N220Q ⁄ N229Q; V >80% is 38 mV) than to activate 80% of the glycosylated Kv3.1s (wild-type Kv3.1; V >80% is 32 mV). Addition- ally, the fraction of channels that were activated as the applied voltage increased was significantly lower for unglycosylated Kv3.1s than for their glycosylated counterparts. The time required for the unglycosylated Kv3.1s to reach their peak current was also less at the various applied potentials relative to the glycosylated Kv3.1s. Activation kinetic values of the partially gly- cosylated Kv3.1s (N229Q and N220Q) appeared to be more similar to those of unglycosylated Kv3.1 than to that of the glycosylated channel. Thus, these results indicate that the occupancy of the N-glycosylation sites is a determining factor for the voltage-dependent acti- vation kinetics of Kv3.1. A recent report on Kv1.1 indicated that N-glycosyla- tion did alter its gating function, and this effect was shown to result from sialic acid residues attached to the oligosaccharide [45]. It was suggested that when the composition of the N-linked oligosaccharide was of a simple type, there was a positive shift in voltage dependence of activation and slower activation kinetics than when the oligosaccharide was of a complex type [45,46]. Therefore, it is possible that the maturation processing of the high-mannose glycan of Kv3.1 to a complex carbohydrate may cause a negative shift in voltage dependence, and an increase in the rate of acti- vation. When the channel activation of wild-type Kv3.1s expressed in Sf9 cells is compared to that expressed both in vivo and in mammalian heterologous expression systems (V >80% is 30 mV; t on at +40 mV is 3.4 ms; t on at +20 mV is 3–4 ms), the voltage dependence of activation appears to be different [31,47]. The results of the aforementioned studies of Kv3.1, along with our report, would suggest that the composition of the N-linked oligosaccharides may influence the voltage dependence of Kv3.1 activation in a similar manner as occupancy of the glycosylation sites. In conclusion, our study indicates that decreases in the occupancy of N-glycosylation sites that may occur in patients suffering from CDGS [15,19] or ER stress- related neurodegenerative diseases [20] could alter the expression of K + currents at the cell surface of neu- rons that express Kv3.1 at high densities. Future stud- ies will be needed to determine whether different compositions of the N-linked oligosaccharide alter the channel activation of Kv3.1. Experimental procedures Materials Spodoptera frugiperda (Sf9) cells were obtained from PharMingen, San Diego, CA, USA. Hink’s TNM-FH insect medium was bought from MediaTech, Inc., Herndan, VA, USA, FBS was from Invitrogen, Carlsbad, CA, USA, and Pluronic F-68 solution, as well as gentamicin, came from Sigma Chemical Co., St Louis, MO, USA. The TA cloning kit and restriction enzymes were acquired from Invitrogen. The BaculoGold transfection kit was purchased from BD Biosciences, San Diego, CA. Plasmid purification columns were obtained from Qiagen, Valencia, CA, USA. Anti-FLAGÒ M2-agarose gel and mouse anti-FLAGÒ M2 were purchased from Sigma, and goat anti-mouse IgG, alkaline phosphatase-conjugated, was purchased from MP Biomedicals, Inc., Irvine, CA, USA. Rabbit anti-Kv3.1b was purchased from Alomone Laboratories, Jerusalem, Israel. Protease inhibitor cocktail set III and Triton X-100 were from CalBiochem, San Diego, CA, USA. Precast SDS ⁄ PAGE gels were procured from Bio-RAD, Hercules, N. L. Brooks et al. Characterization of glycosylation sites in Kv3.1 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS 3295 CA, USA. Ultracentrifuge tubes for SW41 and 70Ti rotors were purchased from Beckman, Palo Alto, CA, USA. All other chemicals used in this study were ordered from Sigma or Fisher Scientific, Co., Hampton, NH, USA. Mutant constructs PCR was used to attach the FLAG sequence (DY- KDDDDK) to the 3¢-end of Rattus rattus Kv3.1 cDNA (accession number P25122), referred to as 3¢FLAG-Kv3.1. The 3¢FLAG-Kv3.1–pCRII recombinant vector was kindly provided by B. Wible and A. M. Brown (Rammelkamp Center for Education and Research, Case Western Univer- sity, Cleveland, OH, USA). The N220Q ⁄ N229Q mutant was constructed by PCR overlap extension [48] using 3¢FLAG-Kv3.1–pCRII as template. Forward and reverse primers were designed to contain nucleotide mismatches that eliminated the two native N-glycosylation sites at posi- tions 220–222 (NKT) and 229–231 (NGT) of Kv3.1 cDNA. PCR products were subcloned into pCR2.1 for amplifica- tion and sequencing. The N220Q ⁄ N229Q was then sub- cloned into EcoRI-digested baculovirus transfer vector, pACSG2. To generate the N220Q and N229Q single mutants, the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) was used (manufacturer’s protocol). Mutagenic forward and reverse primers were designed to contain nucleotide mismatches that eliminated either the first native N-glycosylation site at position 220–222 (NKT) or the second native site at position 229–231 (NGT) of Kv3.1 cDNA, respectively. The dsDNA template was Kv3.1-pacSG2. DNA sequences were verified. Standard procedures were followed for subcloning, and DNA amplification, isolation and sequencing [49]. Cell culture and recombinant baculoviruses Sf9 cells were maintained in Hink’s TNM-FH medium containing 10% FBS, 10 lgÆmL )1 gentamicin, and 0.1% Pluronic F-68 at 27 °C as previously described [50]. Mono- layer Sf9 cultures were used to maintain Sf9 cells and were passaged about twice a week. Suspension cultures of Sf9 cells were seeded from monolayer cultures and stirred at a constant rate of 80–120 r.p.m. Fresh suspension cultures were prepared every 3–5 days. Recombinant baculoviruses were produced by cotransfection of recombinant Baculovi- rus transfer vectors and BaculoGold viral DNA [modified Autographa californica nuclear polyhedrosis virus (AcNPV)]. The manufacturer’s instructions were followed for this procedure (BD Biosciences). Viral seed stocks of intermediate viral titer were generated using monolayer cul- tures. High viral titers were produced in suspension cultures (0.8–0.9 · 10 6 viable cellsÆmL )1 ) using an aliquot of the intermediate viral titer supernatant. Expression of recom- binant proteins required the addition of high viral titer supernatant to a suspension culture (1.1 · 10 6 cellsÆmL )1 ), followed by an incubation period of 24 h at 27 °C. When necessary for studying the occupancy of N-glycosylation sites, TM (25 lgÆmL )1 ) was added to infected cells 15 min postinoculation. Cell fractionation and M2-agarose affinity purification Sf9 cell fractionations were carried out as previously des- cribed [24,25]. Adjustments of the cell fractionation proto- col involved reducing the starting material and the volume of the sucrose layers but maintaining the relative ratios of the sucrose layers. Infected Sf9 cells (about 6.6 · 10 7 ) were harvested by centrifugation at 1204 g in a Beckman SX 4250 rotor for 10 min at 4 °C. The pellet was washed once in 15 mL of ice-cold NaCl ⁄ P i (50 mm Na 2 HPO 4 ; 150 mm NaCl) at pH 7.4, and recentrifuged under the same condi- tions. The pellet was frozen at ) 20 °C for at least 1 h to lyse the cells. Ice-cold homogenizing buffer (250 mm sucrose, 10 mm Tris, pH 7.4; 1 lLÆmL )1 protease inhibitor cocktail set III from Calbiochem; added volume, about 1.8 mL) was used to resuspend the thawed cell pellet. The cells were disrupted in a dounce homogenizer (continuous strokes for at least 10 min) and centrifuged at 500 g for 15 min at 4 °C. All centrifugation was performed using an Eppendorf 45-30-11 rotor, unless otherwise specified. An equal volume of sucrose adjustment buffer (2.55 m sucrose, 10 mm Tris, 1 mm EDTA, pH 7.4) was added to the cleared lysate to increase the sucrose concentration to 1.4 m. The sucrose gradient [2.0 m sucrose, 920 lL; 1.6 m sucrose, 1840 lL; 1.4 m sucrose (sample), about 3680 lL; 1.2 m sucrose, 3680 lL; 0.8 m sucrose, 1840 lL] was pre- pared and centrifuged at 83 472 g in a Beckman SW41 rotor for 2.5 h at 4 °C. Following the centrifugation, plasma membrane (PM), Golgi apparatus (Golgi), and ER fractions were removed and added to ultracentrifuge tubes, where they were diluted by addition of about 10 mL of imi- dazole buffer (25 mm imidazole, 1 mm EDTA, pH 7.4), and centrifuged at 117 734 g in a Beckman 70Ti rotor for 1.5 h to concentrate the fractions. The pellet was solubilized in 200 lL of resuspension buffer (50 mm Na 2 HPO 4 , 0.3 m KCl, pH 7.4, 0.5% Triton X-100) and transferred to micro- centrifuge tubes. The tube was rinsed with 800 lL of pellet resuspension buffer to recover any remaining sample. Next, 20 lL of M2-agarose affinity gel (1 : 1 NaCl ⁄ P i and M2-ag- arose gel slurry) was added to each solubilized sample (total volume, about 1 mL) and incubated on a rotator for 1 h at room temperature. The resin was washed three times with 1 mL of high-K + salt buffer (50 mm Na 2 HPO 4 , 0.3 m KCl, pH 7.4) by centrifugation at 425 g for 3 min. The resin was washed with 1 mL of NaCl ⁄ P i three times by centrifugation (same as previous step), and the washed resin was resus- pended in 100–125 lLof2· SDS ⁄ PAGE sample buffer (62.5 mm Tris, pH 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue) containing 200 mm dithiothreitol. Characterization of glycosylation sites in Kv3.1 N. L. Brooks et al. 3296 FEBS Journal 273 (2006) 3287–3300 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... 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Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism J Physiol 550, 51–66 46 Thornhill WB, Wu MB, Jiang X, Wu X, Morgan PT & Margiotta JF (1996) Expression of Kv1.1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function J Biol Chem 271, 19093–19098 47 Hernandez-Pineda... 2000 g in an Eppendorf F-45-30-11 rotor for 10 min at 4 °C This supernatant was transferred to a clean tube and centrifuged at 100 000 g in a Sorvall TH641 rotor Characterization of glycosylation sites in Kv3.1 for 1 h at 4 °C Pellets from high-speed spins were resuspended in about 4 mL of lysis buffer Protein concentration was measured using a modified Lowry assay A 200 lL aliquot of crude brain membranes . site in the S1–S2 linker of other Kvs (Fig. 1C). For example, the native glycosylation site in the S1–S2 linker of Shaker H4 [40], Kv1 .1 [ 41] , Kv1.2 [42],. SGEY C Fig. 1. Topological model of Kv3. 1 and amino acid sequences of the S1–S2 linker of Kvs. (A) Topology of a Kv3. 1 monomeric unit. Black circles represent the