Amino Acid Properties of Trafficking Determinants in the Outer Pore-Forming Region of Kv1 Potassium Channels in Cell Lines.

Different classes of Kv1 potassium channels have different trafficking patterns despite having very similar amino acid sequences. Two amino acids responsible for these differences have been identified in the outer pore turret region of Kv1.1 and Kv1.4. Here we tested a series of substitutions at these two determinants on Kv1.4. All P506 substitutions tested resulted in a significant decrease in surface protein, total protein, and protein half-life, indicating that proline is required at 506 to stabilize protein conformation and increase trafficking to the cell surface. All K533 substitutions tested had no effect on total protein, suggesting that the lysine at 533 is not important for maintaining Kv1.4 protein conformation. However, a basic or long polar amino acid, such as K, R, or Q, at this position favored high surface protein and efficient trafficking of Kv1.4, whereas an acidic or short amino acid, such as D, E, S, L, N, or H, at this position induced partial high endoplasmic reticulum-retention. This intracellular retention was not due to protein misfolding. We propose that these four prolines and four lysines in a Kv1.4 homotetramer might provide a binding site for a putative endoplasmic reticulum-export molecule to ensure high cell surface protein expression of the channel.

The degree of N-glycosylation affects the trafficking and cell surface expression levels of Kv1.4 potassium channels.

Kv1.4 potassium channels are heavily glycosylated proteins involved in shaping action potentials and in neuronal excitability and plasticity. Kv1.4 N354Q, without an N-glycan, exhibited decreased protein stability and trafficking to the cell surface (Watanabe et al. in J Biol Chem 279:8879-8885, 2004). Here we investigated whether the composition of the N-glycan affected Kv1.4 cell surface expression. Kv1.4 proteins carrying N-glycans with different compositions were generated by adding glycosidase inhibitors or using N-glycosylation-deficient mutant cell lines. We found that oligomannose-type, hybrid-type, or incomplete complex-type N-glycans had a negative effect on surface protein expression of Kv1.4 compared with complex-type N-glycans. The decrease in surface protein level of Kv1.4 was mainly due to a reduction in total protein level, induced by altered N-glycan composition. Kv1.4 in CSTP-treated cells carried a unique oligomannose-type N-glycan that contains three glucose residues. This N-glycan had the most negative effect on cell surface expression of Kv1.4. It decreased Kv1.4 surface protein level by a combined mechanism of reducing total protein level and increasing ER-retention. Our data suggest that composition of the N-glycan plays an important role in protein stability and trafficking, and a sialylated complex-type N-glycan promoted high cell surface expression of Kv1.4.

Antihistamine use in children.

This review provides an overview of the use of antihistamines in children. We discuss types of histamine receptors and their mechanism of action, absorption, onset and duration of action of first-generation and second-generation H(1)-antihistamines, as well as elimination of H(1)-antihistamines which has important implications for dosing in children. The rationale for the use of H(1)-antihistamines is explored for the relief of histamine-mediated symptoms in a variety of allergic conditions including: non-anaphylactic allergic reactions, atopic eczema (AE), allergic rhinitis (AR) and conjunctivitis, chronic spontaneous urticaria (CSU) and whether they have a role in the management of intermittent and chronic cough, anaphylaxis, food protein-induced gastrointestinal allergy and asthma prevention. Second-generation H(1)-antihistamines are preferable to first-generation H(1)-antihistamines in the management of non-anaphylactic allergic reactions, AR, AE and CSU due to: their better safety profile, including minimal cognitive and antimuscarinic side effects and a longer duration of action. We offer some guidance as to the choices of H(1)-antihistamines available currently and their use in specific clinical settings. H(1)-antihistamine class, availability, licensing, age and dosing administration, recommended indications in allergic conditions and modalities of delivery for the 12 more commonly used H(1)-antihistamines in children are also tabulated.

The Kv1.3 potassium channel is localized to the cis-Golgi and Kv1.6 is localized to the endoplasmic reticulum in rat astrocytes.

The functions of voltage-gated potassium (Kv) channels in neurons have been well defined, whereas their roles in glial cells are not fully understood. Kv1.1, Kv1.3 and Kv1.6 are endogenously expressed in C6 astrocytoma cells, but their trafficking and subcellular localization have not been well studied. In C6 cells, Kv1.1 was localized to the cell surface, Kv1.3 was predominantly localized in the cis-Golgi, and Kv1.6 was enriched in the endoplasmic reticulum. Disruption of the Golgi stacks with brefeldin A treatment redirected Kv1.3 to the endoplasmic reticulum, further confirming that Kv1.3 was localized in the Golgi. Denaturing and reducing immunoblot analysis identified an expected Kv1.3 monomer and an unexpected Kv1.3 dimer/aggregate. These two forms had different protein half-lives: that of the monomer form T1/2 was 5.1 h, whereas the dimer/aggregate form was stable over the 8-h measurement period. The Kv1.3 dimer/aggregate form on immunoblots appeared to be correlated with its Golgi retention, based on examination with several cell types that expressed Kv1.3. Glycosidase treatment showed that Kv1.3 contained complex-type N-glycans terminated with sialic acids, suggesting that Kv1.3 had traveled to the trans-Golgi network for sialylation before it was recycled to the cis-Golgi for retention. Inhibition of N-glycosylation did not affect Kv1.3 localization, indicating that N-glycans did not play a role in its Golgi retention. Thus, Kv1.3 appears to be distributed to the cis-Golgi membrane of rat astrocytes in a similar way as a Golgi resident protein, and this unusual distribution appears to be correlated with its SDS/2-mercaptoethanol-resistant dimer/aggregate forms on immunoblots.

Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface.

The voltage-gated potassium channel Kv3.3 is the causative gene of SCA13 (spinocerebellar ataxia type 13), an autosomal dominant neurological disorder. The four dominant mutations identified to date cause Kv3.3 channels to be non-functional or have altered gating properties in Xenopus oocytes. In the present paper, we report that SCA13 mutations affect functional as well as protein expression of Kv3.3 channels in a mammalian cell line. The reduced protein level of SCA13 mutants is caused by a shorter protein half-life, and blocking the ubiquitin-proteasome pathway increases the total protein of SCA13 mutants more than wild-type. SCA13 mutated amino acids are highly conserved, and the side chains of these residues play a critical role in the stable expression of Kv3.3 proteins. In addition, we show that mutant Kv3.3 protein levels could be partially rescued by treatment with the chemical chaperone TMAO (trimethylamine N-oxide) and to a lesser extent with co-expression of Kv3.1b. Thus our results suggest that amino acid side chains of SCA13 positions affect the protein half-life and/or function of Kv3.3, and the adverse effect on protein expression cannot be fully rescued.

Characterization of the Kv1.1 I262T and S342I mutations associated with episodic ataxia 1 with distinct phenotypes.

Episodic ataxia type 1 (EA-1) is an autosomal dominant neurological disorder caused by mutations in the potassium channel Kv1.1. Two EA-1 mutations, I262T and S342I, have been identified with unique clinical phenotypes, but their functional and biochemical properties have not been fully investigated. Here we characterized these two mutations in transfected mammalian cells both electrophysiologically and biochemically. We found that the I262T mutation resulted in a ∼7-fold reduction in the K+ current amplitude compared with wild type channels, whereas the S342I mutation produced an apparent nonfunctional channel when expressed alone. Co-expression of wild type and mutant channels showed that both I262T and S342I exerted dominant-negative effects on wild type function. The protein expression analysis showed that I262T resulted in ∼2-fold decrease in surface protein levels of Kv1.1, which partially contributed to the decreased surface conductance density, whereas the S342I mutation showed no effects on surface protein expression. Conservative amino acid substitution experiments suggest that the wild type amino acids at these two positions are required for normal channel function. Our results broaden the knowledge of EA-1 mutations and the underlying mechanisms of the associated disorder.

N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels.

The voltage-gated potassium channel Kv1.3 plays an essential role in modulating membrane excitability in many cell types. Kv1.3 is a heavily glycosylated membrane protein. Two successive N-glycosylation consensus sites, N228NS and N229ST, are present on the S1-S2 linker of rat Kv1.3. Our data suggest that Kv1.3 contains only one N-glycan and it is predominantly attached to N229 in the S1-S2 extracellular linker. Preventing N-glycosylation of Kv1.3 significantly decreased its surface protein level and surface conductance density level, which were ∼ 49% and ∼ 46% respectively of the level of wild type. Supplementation of N-acetylglucosamine (GlcNAc), l-fucose or N-acetylneuraminic acid to the culture medium promoted Kv1.3 surface protein expression, whereas supplementation of d-glucose, d-mannose or d-galactose did not. Among the three effective monosaccharides/derivatives, adding GlcNAc appeared to reduce sialic acid content and increase the degree of branching in the N-glycan of Kv1.3, suggesting that the N-glycan structure and composition had changed. Furthermore, the cell surface half-life of the Kv1.3 surface protein was increased upon GlcNAc supplementation, indicating that it had decreased internalization. The GlcNAc effect appears to apply mainly to membrane proteins containing complex type N-glycans. Thus, N-glycosylation promotes Kv1.3 cell surface expression; supplementation of GlcNAc increased Kv1.3 surface protein level and decreased its internalization, presumably by a combined effect of decreased branch size and increased branching of the N-glycan.

DmSAS is required for sialic acid biosynthesis in cultured Drosophila third instar larvae CNS neurons.

Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.

The Kv1.2 potassium channel: the position of an N-glycan on the extracellular linkers affects its protein expression and function.

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 conserved position on this linker on Kv1.1-Kv1.5 and Kv1.7 channels. We hypothesize that an N-glycan is found at only this position due to its effect on folding, trafficking, and/or function of these channels. To investigate this hypothesis, N-glycosylation sites were engineered at different positions on the extracellular linkers of Kv1.2 to determine the effects of N-glycans on channel 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 surface protein expression but the N-glycan still affected function similarly as if it were at its native position, (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 and function. We suggest that Kv1 channels have an N-glycan at a conserved position on only the S1-S2 linker to overcome the constraints for proper folding, trafficking, and function that appear to occur if the N-glycan is moved from this position.

Kv1 potassium channel C-terminus constant HRETE region: arginine substitution affects surface protein level and conductance level of subfamily members differentially.

We have shown previously that truncating all of the variable cytoplasmic C-terminus of Kv1.1 potassium channels to G421stop had only a small inhibitory effect on their cell surface conductance density levels and cell surface protein levels. Here we investigated the role of a highly conserved cytoplasmic C-terminal charged region of five amino acids (HRETE) of the S6 transmembrane domain in the protein and conductance expression of Kv1.1, Kv1.2, and Kv1.4 channels. For Kv1.1 we found that E420stop, T419stop, and E418stop showed cell surface conductance densities and cell surface protein levels similar to full length control, whereas R417stop and H416stop exhibited essentially no conductance but their surface protein levels were similar to full length control. A bulky non-negatively charged hydrophilic amino acid at position 417 appeared to be critical for wild type gating of Kv1.1 because R417K and R417Q rescued conductance levels whereas R417A or R417E did not. The R417A mutation in the full length Kv1.1 also exhibited surface protein levels similar to control but it did not exhibit significant conductance. In contrast, mutation of the equivalent arginine to alanine in full length Kv1.2 and Kv1.4 appeared to have little or no effect on channel conductance but rather decreased cell surface protein levels by inducing partial high ER retention. These findings are consistent with the notion that the arginine amino acid in the HRETE region plays a different role in affecting conductance levels or cell surface protein levels of very closely related Kv1 potassium channels.

The glycosylation state of Kv1.2 potassium channels affects trafficking, gating, and simulated action potentials.

We presented evidence previously that decreasing the glycosylation state of the Kv1.1 potassium channel modified its gating by a combined surface potential and a cooperative subunit interaction mechanism and these effects modified simulated action potentials. Here we continued to test the hypothesis that glycosylation affects channel function in a predictable fashion by increasing and decreasing the glycosylation state of Kv1.2 channels. Compared with Kv1.2, increasing the glycosylation state shifted the V(1/2) negatively with a steeper G-V slope, increased activation kinetics with little change in deactivation kinetics or in their voltage-dependence, and decreased the apparent level of C-type inactivation. Decreasing the glycosylation state had essentially the opposite effects and shifted the V(1/2) positively with a shallower G-V slope, decreased activation kinetics (and voltage-dependence), decreased deactivation kinetics, and increased the apparent level of C-type inactivation. Single channel conductance was not affected by the different glycosylation states of Kv1.2 tested here. Hyperpolarized or depolarized shifts in V(1/2) from wild type were apparently due to an increased or decreased level of channel sialylation, respectively. Data and modeling suggested that the changes in activation properties were mostly predictable within and between channels and were consistent with a surface potential mechanism, but those on deactivation properties were not predictable and were more consistent with a conformational mechanism. Moreover the effect on the deactivation process appeared to be channel-type dependent as well as glycosylation-site dependent. The glycosylation state of Kv1.2 also affected action potentials in simulations. In addition, preventing N-glycosylation decreased cell surface Kv1.2 expression levels by approximately 40% primarily by increasing partial endoplasmic reticulum retention and this effect was completely rescued by Kv1.4 subunits, which are glycosylated, but not by cytoplasmic Kvbeta2.1 subunits. The nonglycosylated Kv1.2 protein had a similar protein half-life as the glycosylated protein and appeared to be folded properly. Thus altering the native Kv1.2 glycosylation state affected its trafficking, gating, and simulated action potentials. Differential glycosylation of ion channels could be used by excitable cells to modify cell signaling.

Effects of Kv1.1 channel glycosylation on C-type inactivation and simulated action potentials.

Kv1.1 channels are brain glycoproteins that play an important role in repolarization of action potentials. In previous work, we showed that lack of N-glycosylation, particularly lack of sialylation, of Kv1.1 affected its macroscopic gating properties and slowed activation and C-type inactivation kinetics and produced a depolarized shift in the steady-state activation curve. In our current study, we used single channel analysis to investigate voltage-independent C-type inactivation in both Kv1.1 and Kv1.1N207Q, a glycosylation mutant. Both channels underwent brief and long-lived closures, and the lifetime and frequency of the long-lived closed states were voltage-independent and similar for both channels. We found that, as in macroscopic measurements, Kv1.1N207Q exhibited a approximately 8 mV positive shift in its single channel fractional open time (fo) and a shallower fo-voltage slope compared with Kv1.1. Data suggested that C-type inactivation reflected the equilibration time with at least two slow voltage-independent long-lived closed states that followed the rapid activation process. In addition, data simulation indicated that the C-type inactivation process reflected the equilibration time between the open state and at least two long-lived closed states. Moreover, the faster macroscopic current decay in Kv1.1 mostly reflected a slower equilibration time in these channels as compared with Kv1.1N207Q. Finally, action potential simulations indicated that the N207Q mutation broaden the action potential and decreased the interspike interval. The shape of the action potential was not significantly affected by C-type inactivation, however, for a given channel, C-type inactivation increased the interspike interval. Data and simulations suggested that excitable cells could use differences in K(+) channel glycosylation degree as an additional mechanism to increase channel functional diversity which could modify cell excitability.

Amino acids in the pore region of Kv1 potassium channels dictate cell-surface protein levels: a possible trafficking code in the Kv1 subfamily.

Kv1.1 and Kv1.4 potassium channels have different pore region determinants that were found to affect their cell-surface levels positively and negatively [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419-39427; Zhu, Watanabe, Gomez and Thornhill (2003) J. Biol. Chem. 278, 25558-25567; Zhu, Watanabe, Gomez and Thornhill (2003) Biochem. J. 375, 761-768]. In the present study, we focused on the deep pore region of Kv1 members to test whether a cell-surface trafficking code was dictated by two amino acids. Kv1 channels with a threonine/lysine amino acid pair in a non-contiguous pore region promoted high surface levels, whereas a serine/tyrosine amino acid pair inhibited high surface expression by inducing a high level of partial endoplasmic reticulum retention. Our work suggests that a possible positive trafficking amino acid pair coding here for the Kv1 subfamily is Thr/Lys>Thr/Val>Thr/Tyr>Thr/Arg approximately Thr/His>Ser/Val>Ser/Tyr>Ser/Lys. The Kv1 trafficking code was not transferable to a Kv2 family member and thus it appears that it only governs surface levels in the context of its Kv1 native pore loop region and/or its S5 and S6 regions. All members of a given Kv2, Kv3 or Kv4 potassium channel subfamily have identical amino acids at similar positions in their deep pore regions (Thr/Tyr or Thr/Val), which suggests that any difference in surface levels among members is not dictated by these amino acids. Thus a major determinant for cell-surface trafficking of Kv1 potassium channels is an amino acid pair in their deep pore regions, whereas the cell-surface levels of a given Kv2, Kv3 or Kv4 subfamily member are probably not affected by these amino acids.

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking.

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Allowed N-glycosylation sites on the Kv1.2 potassium channel S1-S2 linker: implications for linker secondary structure and the glycosylation effect on channel function.

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51-66]. Kv1 channel S1-S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1-S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1-S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1-S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1 K+ channels. The S3-S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.

Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic C-terminal VXXSL determinant in regulating cell-surface trafficking.

Kv1.4 and Kv1.1 potassium channel homomers have been shown to exhibit different intracellular trafficking programmes and cell-surface expression levels in cell lines: a determinant in the pore region of Kv1.4 and Kv1.1 [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419-39427] and a cytoplasmic C-terminal VXXSL determinant on Kv1.4 [Li, Takimoto and Levitan (2000) J. Biol. Chem. 275, 11597-11602] have been described, which affected trafficking and cell-surface expression levels. In the present study, we examined whether trafficking pore determinants influenced any cytoplasmic C-terminal trafficking determinant. We found that removal of VXXSL from a Kv1.4 chimaera that contained the pore of Kv1.1 did not affect cell-surface trafficking. Therefore removal of the C-terminal VXXSL of Kv1.4 inhibited protein surface levels only in the presence of the Kv1.4 pore. In contrast, truncating the cytoplasmic C-terminus of Kv1.1 or truncating a Kv1.1 chimaera with the pore of Kv1.4, had little effect on surface protein levels. Furthermore, the subregion of the Kv1.4 pore trafficking determinant that was required for the inhibitory effect of VXXSL removal was mapped to a threonine residue in the deep pore region. Therefore the Kv1.4 pore determinant affected the trafficking and cell-surface levels directed by the C-terminal VXXSL determinant. Different Kv1 trafficking programmes would affect cell-surface expression levels either positively or negatively and also cell signalling. Cells may use differential trafficking programmes of membrane proteins as a post-translational mechanism to regulate surface protein levels and cell function.

Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism.

The effect of glycosylation on Kv1.l potassium channel function was investigated in mammalian cells stably transfected with Kv1.l or Kv1.1N207Q. Macroscopic current analysis showed that both channels were expressed but Kv1.1N207Q, which was not glycosylated, displayed functional differences compared with wild-type, including slowed activation kinetics, a positively shifted V 1/2, a shallower slope for the conductance versus voltage relationship, slowed C-type inactivation kinetics, and a reduced extent of and recovery from C-type inactivation. Kv1. 1N207Q activation properties were also less sensitive to divalent cations compared with those of Kv1.l. These effects were largely due to the lack of trans-Golgi added sugars, such as galactose and sialic acid, to the N207 carbohydrate tree. No apparent change in ionic current deactivation kinetics was detected inKv1.1N207Q compared with wild-type. Our data, coupled with modelling, suggested that removal of the N207 carbohydrate tree had two major effects. The first effect slowed the concerted channel transition from the last dosed state to the open state without changing the voltage dependence of its kinetics. This effect contributed to the G-V curve depolarization shift and together with the lower sensitivity to divalent cations suggested that the carbohydrate tree and its negatively charged sialic acids affected the negative surface charge density on the channel's extracellular face that was sensed by the activation gating machinery. The second effect reduced a cooperativity factor that slowed the transition from the open state to the dosed state without changing its voltage dependence. This effect accounted for the shallower G-V slope, and contributed to the depolarized G-V shift, and together with the inactivation changes it suggested that the carbohydrate tree also affected channel conformations. Thus N-glycosylation, and particularly terminal sialylation, affected Kv1.l gating properties both by altering the surface potential sensed by the channel's activation gating machinery and by modifying conformational changes regulating cooperative subunit interactions during activation and inactivation. Differences in glycosylation pattern among closely related channels may contribute to their functional differences and affect their physiological roles.

The permeation and activation properties of brain sodium channels change during development.

BTX-modified sodium channels from 15-day embryonic (E15) rat forebrains were studied in planar lipid bilayers. Compared to postnatal sodium channels, E15 channels had a lower maximal single channel conductance, whereas their permeation pathway sensed a comparable surface charge density and had a similar apparent binding affinity for sodium ions. The steady-state activation curve of E15 channels was significantly more hyperpolarized and had a shallower slope than postnatal channels. The apparent BTX binding affinity was significantly lower for E15 channels than for postnatal channels. Finally, E15 channel alpha-subunits displayed a lower apparent molecular weight, and a lower sialylation level than postnatal sodium channel alpha-subunits. Together with previous studies, our data suggested that the observed functional differences between E15 and postnatal voltage-dependent sodium channels cannot be explained solely by the observed differences in channel sialylation, and hence they also appeared to reflect the presence of other channel structural differences.

Heteromeric Kv1 potassium channel expression: amino acid determinants involved in processing and trafficking to the cell surface.

Kv1.4 and Kv1.1 potassium channels are expressed in brain as mature glycoproteins that are trans-Golgi glycosylated. When expressed in cell lines these homomers had very different trans-Golgi glycosylation efficiencies and cell surface expression levels with Kv1.4 > Kv1.1 for both parameters (Zhu, J., Watanabe, I., Gomez, B., and Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419-39427). This previous study identified determinants in the outer pore region of Kv1.4 and Kv1.1 that positively and negatively, respectively, affected these events when expressed as homomers. Here we investigated which subunit exhibited positive or negative effects on these processes when expressed as heteromers. Kv1.4/Kv1.1 heteromers, by coexpression or expression as tandem-linked heteromers, were expressed on the cell surface at approximately 20-fold lower levels versus Kv1.4 homomers but they were trans-Golgi glycosylated. The lower Kv1.4/Kv1.1 expression level was not rescued by Kvbeta 2.1 subunits. Thus Kv1.1 inhibited high cell surface expression and partially retained the heteromer in the endoplasmic reticulum, whereas Kv1.4 stimulated trans-Golgi glycosylation. The subunit determinants and cellular events responsible for these differences were investigated. In a Kv1.4/Kv1.1 heteromer, the Kv1.1 pore was a major negative determinant, and it inhibited high cell surface expression because it induced high partial endoplasmic reticulum retention and it decreased protein stability. Other Kv1.1 regions also inhibited high surface expression of heteromers. The Kv1.1 C terminus induced partial Golgi retention and contributed to a decreased protein stability, whereas the Kv1.1 N terminus contributed to only a decreased protein stability. Thus a neuron may regulate its cell surface K+ channel protein levels by different Kv1 subfamily homomeric and heteromeric combinations that affect intracellular retention characteristics and protein stability.