谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

理性代谢工程改造促进谷氨酸棒杆菌高效合成L-谷氨酸

L-谷氨酸是世界上第一大宗氨基酸产品,广泛应用于食品医药及化工等行业。以谷氨酸高产菌谷氨酸棒杆菌(Corynebacterium glutamicum) G01为出发菌株,首先通过敲除主要副产物丙氨酸合成相关基因-丙氨酸氨基转移酶编码基因(alaT),降低了发酵副产物丙氨酸含量。其次,α-酮戊二酸节点碳流量对谷氨酸合成起重要作用,因此,采用核糖体结合位点(ribosome-binding site,RBS)序列优化降低了α-酮戊二酸脱氢酶的活性,强化了谷氨酸合成代谢流。同时通过筛选不同来源的谷氨酸脱氢酶,加强了α-酮戊二酸内源转化为谷氨酸的能力。接着,对谷氨酸转运蛋白进行理性设计,提高了谷氨酸的外排能力。最后,对基于以上策略构建的整合菌株进行了5 L发酵罐发酵优化,通过梯度升温结合分批补料策略,谷氨酸产量为(136.33±4.68) g/L,较原始菌的产量(96.53±2.32) g/L提高了41.2%;糖酸转化率为55.8%,较原始菌的44.2%提高了11.6%;且降低了副产物丙氨酸的含量。以上策略一定程度上提高了谷氨酸的产量与糖酸转化率,可为谷氨酸生产菌株的代谢改造提供参考。     

TCA循环关键节点对L-谷氨酸合成的影响

谷氨酸是一种重要的氨基酸,其衍生出来的高值化产品具有广泛的应用,市场需求量巨大。文中通过对出发菌株谷氨酸棒杆菌Corynebacterium glutamicum E01和谷氨酸高产菌C. glutamicum G01进行转录组测序与重测序分析,挑选中心代谢途径中转录水平和基因水平上存在差异的基因进行研究,以挖掘出对谷氨酸合成影响较大的基因进一步提高谷氨酸的产量。草酰乙酸节点和α-酮戊二酸节点在谷氨酸合成中扮演着重要角色,探索研究了草酰乙酸节点和α-酮戊二酸节点对谷氨酸生产的扰动影响。综合以上实验结果构建的整合菌株,5 L发酵罐发酵过程中其菌体生长速率较原始菌略有降低,但48h的谷氨酸产量高达(136.09±5.53)g/L,较原始菌的(93.53±4.52)g/L提高了45.5%;糖酸转化率提高至58.9%,较原始菌的45.2%提高了13.7%。可见,上述实验策略的应用在一定程度上提高了谷氨酸产量和糖酸转化率,为谷氨酸棒杆菌的代谢工程改造提供了理论基础。     

植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。     

谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。     

代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸

5-氨基乙酰丙酸 (5-aminolevulinic acid,5-ALA) 在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成。为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造。首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸合成酶ALAS,选择酶活相对较高的沼泽红假单胞菌的RphemA基因作为关键合成酶基因,并筛选到能显著增强RphemA的酶活性的核糖体结合位点RBS5。重组菌株ALAS的比酶活可达 (221.87±3.10) U/mg,且5-ALA产量提高了14.3%;随后通过敲除α-酮戊二酸脱氢酶抑制蛋白基因 (odhI) 和琥珀酸脱氢酶基因 (sdhA),促进了前体琥珀酰CoA向5-ALA途径的流动;通过sRNA抑制hemB表达减少了5-ALA的降解;并且过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了5-ALA的输出效率;使用重组菌株C. glutamicum 13032/?odhI/?sdhA-sRNAhemB-RBS5RphemA-eamA摇瓶发酵,5-ALA最高产量达11.90 g/L,较出发菌株提高了57%。最后,在5 L发酵罐中进行补料分批发酵,48 h内5-ALA的产量达25.05 g/L,为目前以葡萄糖为碳源发酵的最高产量。本研究构建了高产5-ALA重组谷氨酸棒杆菌,具有良好的工业应用前景。     

代谢木糖重组谷氨酸棒杆菌的构建

为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。     

胃(H++K+)-ATPase结构与功能的研究

胃(H⁺+K⁺)-ATPase属于生物膜的第二类质子泵(E₁E₂型),从生理角度它是胃酸分泌的质子泵。本文结合我们初步的研究结果:猪、大白鼠胃粘膜(H⁺+K⁺)-ATPase的纯化以及由消炎痛引起的急性胃粘膜病变与胃粘膜(H⁺+K⁺)-ATPase的关系等,对此酶在近十几年来它的纯化、结构、性质、催化机理,向胃腔分泌盐酸的功能及其调节和胃病变的分子机理等方面进行了简要的综述。     

谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除

本研究以谷氨酸棒杆菌（Corynebacterium glutamicum）标准菌株ATCC 13032染色体为模板,设计引物PCR扩增高丝氨酸脱氢酶编码基因（hom）,在hom基因内部插入一段来源于质粒pET28a的卡那霉素抗性基因（Km）,得到基因元件hom::Km;通过电击转化法将hom::Km转入出发菌株替换原菌株的hom,在含卡那霉素的平板上挑取阳性转化子,通过PCR验证得到高丝氨酸脱氢酶缺陷的重组菌。发酵结果表明重组菌C.g- hom::Km -8发酵60小时赖氨酸产量达到4.7 g／L,是出发菌株谷氨酸棒杆菌ATCC 13032（0.7 g／L）的6.7倍。     

谷氨酸棒杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种具有重要应用价值的四碳平台化合物。微生物法发酵生产琥珀酸以其社会、环境和经济优势展现出良好的发展前景。谷氨酸棒杆菌被广泛应用于氨基酸、核苷酸等高附加值化学品的工业化生产,在厌氧条件下细胞处于生长停滞状态,但仍能高效利用碳源合成有机酸,通过代谢工程改造的谷氨酸棒杆菌有望成为理想的琥珀酸生产菌株。结合近年来谷氨酸棒杆菌生产琥珀酸取得的最新成果,本文综述了构建高产琥珀酸工程菌株的代谢工程策略、底物的扩展利用,并展望了将来的研究方向。     

Introduction of a carboxyl group in the loop of the F0 c-subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803

The proton translocation stoichiometry (H⁺/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H⁺/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (G _p ) and the proton gradient ( _H ⁺) induced by acid–base transition. The mutant displayed a significantly higher H⁺/ATP ratio than the control strain (wild type with kanamycin resistance) at pH 8 (4.3 vs. 3.3); the higher ratio also being observed in chloroplasts and Synechococcus 6716. Furthermore, the pH dependence of the H⁺/ATP of strain plc37 resembles that of Synechococcus 6716. When the pH was increased from 7.6 to 8.4, the H⁺/ATP of the mutant increased from 4.2 to 4.6 whereas in the control strain the ratio decreased from 3.8 to 2.8. Differences in H⁺/ATP between the mutant and the control strain were confirmed by measuring the light-induced phosphorylation efficiency (P/2e), which changed as expected, i.e., the P/2e ratio in the mutant was significantly less than that in the wild type. The need for more H⁺ ions used per ATP in the mutant was also reflected by the significantly lower growth rate of the mutant strain. The results are discussed against the background of the present structural and functional models of proton translocation coupled to catalytic activity of the ATP synthase.     

Enhanced glutamic acid production by a H+-ATPase-defective mutant of Corynebacterium glutamicum

Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 microg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 microg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 microg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.     

Effect of glutamic acid oversynthesis on the development of cyanide-resistant respiration in the bacterium Corynebacterium glutamicum

The composition of the respiratory chains of the wild stain Corynebacterium glutamicum and of its mutant differing in their ability for the glutamic acid oversynthesis in a medium with melassa was studied. Under excess of biotine and the parent strain is incapable of acid oversynthesis, while the mutant forms and excretes the acid. Both bacterial strains contain menaquinone and equal sets of cytochromes C550, b556, b563, and a600. The membrane-bound dehydrogenases of the parent strain are represented by NADH-, NADPH- and succinate dehydrogenases. Unlike the parent strain, the mutant membrane preparation does not oxidize NADPH. Both strains do not practically differ in their menaquinone content. The cyanide-resistant oxidase of a non-cytochrome nature appears in the wild strain during its transfer to the stationary growth phase. Induction of glutamic acid oversnythesis by addition of penicilline prevents the formation of the cyanide-resistant oxidase. On the contrary, the mutant transfer to the stationary growth phase is not accompanied by a formation of cyanide-resistant oxidase, which appears only after cessation of glutamic acid oversynthesis. Induction of the cyanide-resistant respiration by addition of cyanide inhibits the acid oversynthesis. Oxidation of substrates by membrane preparations of both bacterial strains in the absence and presence of cyanide is not followed by the hydrogen peroxide formation. It is assumed that there exist competitive interactions between the supersynthesis of glutamic acid and the cyanide-resistant respiration. The possible structure of the respiratory chain of Cor. glutamicum is discussed.     

Functional Analysis of the Na+/H+ Antiporter Encoding Genes of the Cyanobacterium Synechocystis PCC 6803

The role of putative Na⁺/H⁺ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na⁺/H⁺ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na⁺/H⁺ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na⁺/H⁺ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na⁺/H⁺ antiporters was studied. No induction of any Na⁺/H⁺ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na⁺/H⁺ antiporter encoding genes showed induction. These results might indicate that some of Na⁺/H⁺ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.     

Effect of Reduced H+-ATPase Activity on Acid Tolerance in Streptococcus bovis Mutants

In order to confirm that H⁺-ATPase plays an important role in the acid tolerance ofStreptococcus bovis , two mutants with low activities of H⁺-ATPase were isolated by use of ethyl methanesulfonate and neomycin resistance. The activity of H⁺-ATPase per cellular nitrogen was related to the lowest culture pH permitting growth. A mutant with little H⁺-ATPase activity (Mutant 2) was unable to grow below pH 5.5, which suggests that the intracellular pH should be maintained above 5.5 in S. bovis. Since lactate dehydrogenase activity, which is important for acid tolerance, was similar in parent and mutant strains, H⁺-ATPase activity is likely to affect acid tolerance. The amount of H⁺-ATPase protein as determined by Western-blot analysis with polyclonal antibody, was similar in Mutant 2 and its parent, indicating that H⁺-ATPase activity per enzyme protein is reduced by mutation. Probably, H⁺-ATPase synthesis was not changed by mutation. The gene encoding H⁺-ATPase of Mutant 2 had mutations at positions close to the ATP-binding motif A sequence in the β-subunit, which probably explains the reduced activity of H⁺-ATPase in this mutant. These results strongly support the assumption that H⁺-ATPase has a key role in the acid tolerance of S. bovis.     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

【目的】探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能。【方法】使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使丙酮丁醇梭菌稳定地在产酸期和产溶剂期生长,分析野生型菌株和突变株在稳定的产酸期和产醇期的生长和产物合成情况;通过添加不同浓度的过氧化氢检测野生型和突变株的抗氧化压力。【结果】抗性筛选和基因测序结果表明,成功构建了硫氧还蛋白还原酶失活的突变株,命名为Clostridiumacetobutylicum trxB::int(29)。在分批发酵中,突变株和野生型菌株的最大生长量相近,细胞在600 nm处的光吸收值(OD600)达到6.5,但是突变株在36 h的OD600达到最大,较野生型推迟12 h;在连续发酵的产酸期,野生型菌株与突变株生长变化不大,OD600分别稳定在4.6和4.4,且葡萄糖的消耗和酸产量相差不大;在产溶剂期,突变株的OD600稳定在3.5,低于野生型的4.0...     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).