PubMed Health⌕ Search

Biomedical subjects

Itaru Watanabe

Publications and source records attributed to Itaru Watanabe.

11 recordsLinked to original sources
PubMed2005

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.

Action Potentials↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

Detection of Leuconostoc strains at a meat processing plant using polymerase chain reaction.

To simplify the labor-intensive conventional routine testing of samples to detect Leuconostoc at a meat processing plant, we developed polymerase chain reaction (PCR) primers specific for Leuconostoc from 16S rRNA gene sequences. These primers did not detect other common lactic acid bacteria such as Lactobacillus plantarum, Lact. sake, Lact. fermentum, Lact. acidophilus and Weissella viridescens. PCR with this primer detected all Leuconostoc species tested (Leu. mesenteroides subsp. mesenteroides, Leu. pseudomesenteroides, Leu. carnosum, Leu. lactic, Leu. citreum, Leu. amelibiosum, Leu. gelidum), except for Leu. fallax, and no other lactic acid bacteria on agarose gel electrophoresis. The method could identify areas contaminated with Leuconostoc in a large-scale industrial meat processing plant. Of 69 samples analyzed, 34 were positive for Leuconostoc according to the conventional culture method (isolation of LAB producing dextran) and PCR, whereas 29 were negative according to both. Six samples were culture-negative but positive by PCR. No false negative results were generated by PCR. The method is rapid and simple, is useful for routinely monitoring areas contaminated with Leuconostoc in meat processing plants, and could help to prevent the spoilage of meat products.

DNA Primers↗
PubMed2004

Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis.

We established a novel system using denaturing gradient gel electrophoresis (DGGE) to quickly identify bacteria known to be responsible for spoilage in meat processing plants and meat products. We extracted bacterial DNA from swabbed samples at various locations in the plant and from meat products and performed PCR amplification, targeting 16S rDNA from the dominant organisms. The amplification products were subjected to DGGE, and the contaminating bacteria in the meat products and the plant were analyzed. This analysis indicated that lactic acid bacteria and spoilage-causing bacteria are widely distributed within the meat processing plant. We developed molecular size markers to identify the dominant organisms obtained from the plant and meat products. The establishment of the present method allows quick and simple identification of bacteria causing the possible deterioration of products and contamination and thus permits constant monitoring of any harmful bacteria within meat processing plants.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2003

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.

Amino Acid Motifs↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2002

Short-term (-)-hydroxycitrate ingestion increases fat oxidation during exercise in athletes.

(-)-Hydroxycitrate (HCA) is known to inhibit increasing malonyl CoA concentration during endurance exercise. Furthermore, a short-term administration of HCA enhances endurance exercise performance in mice. Therefore we investigated the short-term administration of HCA on the exercise performance of athletes. Subjects were administered 250 mg of HCA or placebo as a control (CON) for 5 d, after each time performing cycle ergometer exercise at 60% VO2max for 60 min followed by 80% VO2max until exhaustion. Blood was collected and expired gas samples analyzed at rest and every 15 min. The respiratory exchange ratio was significantly lower in the HCA trial than in the CON trial (p < 0.05). Fat oxidation was significantly increased by short-term administration of HCA, and carbohydrate oxidation was significantly decreased (p < 0.05) during exercise, presumably resulting in increasing the cycle ergometer exercise time to exhaustion after 1 h of 60% VO2max exercise (p < 0.05). These results suggest that a short-term administration of HCA enhances endurance performance with increasing fat oxidation, which spares glycogen utilization during moderate intensity exercise in athletes.

Adipose Tissue↗