How Does the Plant Plasma Membrane H+-ATPase Pump Protons?

The plasma membrane H⁺-ATPase couples ATP hydrolysis to protontransport thereby establishing the driving force for solutetransport into and out of plant cells. As such, this enzymeparticipates in a number of cellular processes important tothe overall physiology of plants. From biochemical studies andthe recent application of molecular approaches, the enzyme reactionmechanism and structure of this protein have been characterized.However, our basic understanding of how this enzyme links theendergonic reaction of ATP hydrolysis to proton translocationis far from complete. In this review, several significant questionsregarding the energy coupling mechanism will be addressed interms of information available on the plant plasma membraneH⁺-ATPase and from studies on other P-type transport ATPases.These questions focus on the chemical nature of proton translocation,how this is linked or driven by the ATP hydrolysis reactionand what role, if any, K⁺ has in the transport process. Key words: Energy coupling, membranes, bioenergetics, ion transport, P-type transport ATPase     

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso-PC) on ATP and ρ-nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H+-ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso-PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso-PC was within 0-0.03%, and increased slightly when lyso-PC was higher than 0.03%. At the concentration of 0.03%, lyso-PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46μmol Pi·mg-1 protein·min-1 to 0.87 μmol Pi·mg-1 protein·min-1 while Km increased from 0.88 mmol/L to 1.15 mmol/L under lyso-PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0. Moreover, it was found lyso-PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso-PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso-PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C-terminal autoinhibitory domain in the plant plasma membrane H+-ATPase.     

Changes in Inhibitory Effect of Vanadate on the Plasma Membrane H+-ATPase Under Trypsin Treatment

The changes in inhibitory effect of vanadate on the plasma membrane H + ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean （Glycine max L.）hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%,while that of the un-cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the Km values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, Km remained as 0.34 mmol/L,while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C-terminal end of the plasma membrane H+ ATPase.     

Influence of K＋ on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H＋-ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean ( Glycine max L. ) hypocotyls by two-phase partitioning methods. The stimulatory effects of K+ on the coupling between ATP hydrolysis and proton transport by the plasma membrane H+-ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KC1, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that Km of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while Vmax of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg- l protein·min-1 in the presence of KC1. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KC1, respectively. Further studies revealed that K+ could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K+ could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H+ -ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H + -ATPase.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Effect of Activators and Blockers of Ligand-Regulated Ion Channels on the Activity of the Cl−-Stimulated Mg2+-ATPase of the Plasma Membrane Fraction from Bream (Abramis brama L.) Brain

Effects of GABA, glycine, acetylcholine, and glutamate (agonists of the GABA_a/benzodiazepine, glycine, choline, and glutamate receptors, respectively) at concentrations in the range 10^–8-10^–4 M on the activity of basal Mg²⁺-ATPase of the plasma membrane fraction from bream brain and on its activation by Cl^– were investigated. GABA and glycine activated basal Mg²⁺-ATPase activity and suppressed its activation by Cl^–. Acetylcholine and glutamate activated basal Mg²⁺-ATPase to a lesser extent and did not suppress the activation of the enzyme by Cl^–.The activation of basal Mg²⁺-ATPase by neuromediators was decreased by blockers of the corresponding receptors (picrotoxin, strychnine, benztropine mesylate, and D-2-amino-5-phosphonovaleric acid). In addition, picrotoxin and strychnine eliminated the inhibiting effect of GABA and glycine, respectively, on the Cl^–-stimulated Mg²⁺-ATPase activity. Agonists of the GABA_a/benzodiazepine receptor–phenazepam (10^–8-10^–4 M) and pentobarbital (10^–6-10^–3 M)–activated the basal Mg²⁺-ATPase activity and decreased the Cl^–-stimulated Mg²⁺-ATPase activity. The dependence of both enzyme activities on ligand concentration is bell-shaped. Moreover, phenazepam and pentobarbital increased the basal Mg²⁺-ATPase activity in the presence of 10^–7 M GABA and did not influence it in the presence of 10^–4 M GABA and 10^–6 M glycine. The data suggest that in the fish brain membranes the Cl^–-stimulated Mg²⁺-ATPase interacts with GABA_a/benzodiazepine and glycine receptors but not with m-choline and glutamate receptors.     

Importin α Partitioning to the Plasma Membrane Regulates Intracellular Scaling

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A Membrane-proximal,C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β₂ adrenergic receptor (β₂AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β₂AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β₂AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.     

植物质膜H+-ATPase的研究进展

质膜H -ATPase参与植物细胞的物质跨膜转运、细胞的伸长生长、气孔的开闭以及植物对环境胁迫的响应等生理过程,是植物生命活动的“主宰酶”。其活性调节涉及激素、环境因子等多种因素,可发生在转录、翻译和酶分子等多级水平。因此,在植物生长发育过程中,质膜H -ATPase活性的调节对生理活动起重要作用。本文就植物质膜H -ATPase的结构特征、生理功能、活性变化及其调节机理等的研究进展进行综述,以进一步揭示该酶的生理功能及其调节机理与植物生命活动过程的关系。     

Combined Effect of Diazepam and GABAA-ergic Ligands on the Activity of Cl–-ATPase in Plasma Membrane of Bream Brain (Abramis brama L.)

We studied the combined effect of diazepam and GABA_A-ergic ligands on the activity of Cl^–-ATPase in plasma membrane of bream brain. The membrane fraction were preincubated and incubated with diazepam as well as with other GABA_A-ergic ligands at physiological pH (7.4), i.e. under the conditions when Cl^–-ATPase activity is undetectable. GABA (0.1–100 M) induced Cl^–-ATPase activity with the maximum effect at 10 M. Diazepam (0.1 M) enhanced the effect of low GABA concentrations (0.1–1 M) on Cl^–-ATPase activity but had no effect on the enzyme in the presence of high GABA concentrations (10–100 M). At the same time, GABA (1 M) enhanced the effect of low diazepam concentrations (0.1–1 M) on the enzyme activity but had no effect on it in the presence of high concentrations of the ligand. Blockers of GABA_A-ergic receptors, picrotoxin (50 M) and bicuculline (5 M), canceled the combined effect of diazepam and GABA on the enzyme activity. The obtained data demonstrate that the combined effect of diazepam and GABA_A-ergic ligands on Cl^–-ATPase activity at physiological pH is similar to the effect of these ligands on GABA_A/benzodiazepine/Cl^– channel.     

植物细胞质膜H+-ATPase的调控

综述了质膜H ATPase在转录、翻译及翻译后水平上所受调控现象及其机制的研究进展     

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO 3 − in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na⁺/H⁺ and H⁺-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na⁺/H⁺ exchange activity (assayed by ²²Na⁺ influx) or abundance (using NHE-3 specific antibody) and H⁺-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na⁺/ H⁺ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO ₃ ^– 3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO ₃ ^– 90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H⁺-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na⁺/H⁺ exchanger activity increased by 29% in high HCO ₃ ^– group (HCO ₃ ^– 96 mm) and decreased by 16% in the low HCO ₃ ^– groups (HCO ₃ ^– 7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO ₃ ^– ] tubules. The abundance of H⁺-ATPase, however, increased by 82% in high [HCO ₃ ^– ] and decreased by 77% in the low [HCO ₃ ^– ] tubules. In PT suspensions incubated in varying pCO₂ and constant [HCO ₃ ^– ], Na⁺/H⁺ exchanger activity increased by 35% in high pCO₂ (20% pCO₂, respiratory acidosis) and decreased by 32% in low pCO₂ (1.5% pCO₂, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO₂ tubules. However, the H⁺-ATPase abundance increased by 74% in high pCO₂ and decreased by 69% in low pCO₂ tubules.The results of these studies suggest that the luminal Na⁺/H⁺ exchanger is predominantly regulated by pH whereas H⁺-ATPase is mainly regulated by [HCO ₃ ^– ] and/ or pCO₂. They further suggest that the adaptive changes in H⁺-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na⁺/H⁺ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)These studies were supported by a Merit Review Grant from the Department of Veterans Affairs and a grant-in-aid from the American Heart Association (to M.S.), a Baxter Health Care Grant (to B.B.), and the National Institute of Health Grants DK 38510 (to E.B.C. and M.C.R.) and DK 42086 (to E.B.C.).     

Cell pH and H+ Secretion by S3 Segment of Mammalian Kidney: Role of H+-ATPase and Cl−

The role of H⁺-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na⁺- and Cl⁻-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl⁻ channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na⁺ solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min⁻¹, not significantly different from zero (P > 0.42). For superfusion with Na⁺0 Ringer, this variation was 0.081 ± 0.015 min⁻¹, P < 0.001 against 0. These slopes were markedly reduced by the Cl⁻ channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na⁺-free medium is probably due to a vacuolar H⁺-ATPase, whose activity is stimulated in the presence of Cl⁻, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl⁻ and on the integrity of the cytoskeleton. Received: 11 April 2000/Revised: 14 August 2000     

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2)

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.     

Leukocyte Adhesion—an Integrated Molecular Process at the Leukocyte Plasma Membrane

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell–cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific ₂-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin–intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

The Tetanus Neurotoxin‐Sensitive and Insensitive Routes to and from the Plasma Membrane: Fast and Slow Pathways?

Intracellular membrane trafficking in eukaryotes involves the budding of vesicles from a donor compartment, their translocation, and subsequent fusion with a target membrane. This last step has been shown to involve SNARE proteins, classified into two categories, vesicular (v)-SNAREs and target (t)-SNAREs. It is the pairing of v- and t-SNAREs that is responsible for bringing the lipid bilayers together for membrane fusion. Key to the discovery of SNAREs is the sensitivity of their neuronal synaptic prototypes, which mediate the release of neurotransmitters, to clostridial neurotoxins. In this review, we focus on tetanus neurotoxin-sensitive and tetanus neurotoxin-insensitive v-SNAREs, in particular synaptobrevin and cellubrevin, both tetanus neurotoxin-sensitive and Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, also called VAMP7). The brevins are characterized by an RD sequence in the middle of their SNARE motif whereas TI-VAMP has an RG sequence. These two categories of exocytic v-SNAREs define two important routes to and from the plasma membrane: one sensitive, the other insensitive to tetanus neurotoxin. We also discuss the central role of the endosomal system that could be considered, as already suggested for Rab proteins, as a mosaic of v-SNAREs, thus raising the question of whether or not these two routes can merge, and if so, how and where.     

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding

We have isolated the plasma membrane H⁺−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H⁺-ATPase, thus activating H⁺ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H⁺-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase.     

Effect of Naloxone on the Activity of Cl–-Activated Mg2+-ATPase from Plasma Membrane Fraction of Bream Brain (Abramis brama L.) in the Presence of GABAa-ergic Substances

We studied the effect of naloxone—an antagonist of the opioid receptors—on sensitivity of Cl^–-activated Mg²⁺-ATPase from the plasma membrane fraction of bream brain (Abramis brama L.) to GABA_a-ergic substances. Preincubation of the plasma membranes with 1–100 M naloxone increased the basal Mg²⁺-ATPase activity and suppressed its activation by chloride ions. The same effects were observed in the presence of the agonists of GABA_a/benzodiazepine receptors: 0.1–100 M GABA, 1–500 M pentobarbital, and 0.1–100 M phenazepam. Naloxone (10 M) inhibited activation of the basal Mg²⁺-ATPase by the studied ligands and restored the enzyme sensitivity to Cl^–. However, the effect of naloxone was not observed in the presence of high concentrations of pentobarbital (500 M) and phenazepam (100 M). The obtained data show that naloxone modulates the activity of Cl^–-activated Mg²⁺-ATPase from the plasma membranes of bream brain and antagonizes the GABA_a receptor ligands.     

The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e-subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

The β-subunit associates with the α₁ pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β_2a and β_2e, associate with the plasma membrane in the absence of the α₁-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β_2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β_2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β_2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β_1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β_2e, but not with a β_2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β_2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.