Cloning and characterization of mitochondrial methionyl-tRNA synthetase from a pathogenic fungi Candida albicans

A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNA^Met was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNA^Met and MetRS.     

Membrane structure of CtrA3, a copper-transporting P-type-ATPase from Aquifex aeolicus

We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting P_IB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. We have cloned both copper pumps, expressed them in Escherichia coli and characterized them functionally. CtrA2 is activated by Ag⁺ and Cu⁺ and presumably transports reduced Cu⁺, while CtrA3 is activated by, and presumably transports, the oxidized copper ion. Both CtrA2 and CtrA3 are thermophilic proteins with an activity maximum at 75 °C. Electron cryomicroscopy of two-dimensional crystals of CtrA3 yielded a projection map at ∼7 Å resolution with density peaks, indicating eight membrane-spanning α-helices per monomer. A fit of the Ca-ATPase structure to the projection map indicates that the arrangement of the six central helices surrounding the ion-binding site in the membrane is conserved, and suggests the position of the two additional N-terminal transmembrane helices that are characteristic of the heavy metal, eight-helix P_1B-type ATPases.     

Characterization of Schizosaccharomyces pombe Copper Transporter Proteins in Meiotic and Sporulating Cells

Meiosis requires copper to undertake its program in which haploid gametes are produced from diploid precursor cells. In Schizosaccharomyces pombe, copper is transported by three members of the copper transporter (Ctr) family, namely Ctr4, Ctr5, and Ctr6. Although central for sexual differentiation, very little is known about the expression profile, cellular localization, and physiological contribution of the Ctr proteins during meiosis. Analysis of gene expression of ctr4⁺ and ctr5⁺ revealed that they are primarily expressed in early meiosis under low copper conditions. In the case of ctr6⁺, its expression is broader, being detected throughout the entire meiotic process with an increase during middle- and late-phase meiosis. Whereas the expression of ctr4⁺ and ctr5⁺ is exclusively dependent on the presence of Cuf1, ctr6⁺ gene expression relies on two distinct regulators, Cuf1 and Mei4. Ctr4 and Ctr5 proteins co-localize at the plasma membrane shortly after meiotic induction, whereas Ctr6 is located on the membrane of vacuoles. After meiotic divisions, Ctr4 and Ctr5 disappear from the cell surface, whereas Ctr6 undergoes an intracellular re-location to co-localize with the forespore membrane. Under copper-limiting conditions, disruption of ctr4⁺ and ctr6⁺ results in altered SOD1 activity, whereas these mutant cells exhibit substantially decreased levels of CAO activity mostly in early- and middle-phase meiosis. Collectively, these results emphasize the notion that Ctr proteins exhibit differential expression, localization, and contribution in delivering copper to SOD1 and Cao1 proteins during meiosis.     

Molecular and functional analyses of COPT/Ctr-type copper transporter-like gene family in rice

Background  

The copper (Cu) transporter (COPT/Ctr) gene family has an important role in the maintenance of Cu homeostasis in different species. The rice COPT-type gene family consists of seven members (COPT1 to COPT7). However, only two, COPT1 and COPT5, have been characterized for their functions in Cu transport.     

Ctr1 Is an Apical Copper Transporter in Mammalian Intestinal Epithelial Cells in Vivo That Is Controlled at the Level of Protein Stability

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu⁺ importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.     

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency

Copper is an essential micronutrient that functions as a redox cofactor in multiple plant processes, including photosynthesis. Arabidopsis thaliana possesses a conserved family of CTR-like high-affinity copper transport proteins denoted as COPT1-5. COPT1, the only family member that is functionally characterized, participates in plant copper acquisition. However, little is known about the function of the other Arabidopsis COPT proteins in the transport and distribution of copper. Here, we show that a functional fusion of COPT5 to the green fluorescent protein localizes in Arabidopsis cells to the prevacuolar compartment. Plants defective in COPT5 do not exhibit any significant phenotype under copper-sufficient conditions, but their growth is compromised under copper limitation. Under extreme copper deficiency, two independent copt5 knockout mutant lines exhibit severe defects in vegetative growth and root elongation, low chlorophyll content, and impairment in the photosynthetic electron transfer. All these phenotypes are rescued when the wild-type copy of the COPT5 gene is retransformed into a copt5 knockout line or when copper, but not other metals, are added to the medium. COPT5 is expressed in vascular tissues, with elevated levels in roots. Taken together, these results suggest that COPT5 plays an important role in the plant response to environmental copper scarcity, probably by remobilizing copper from prevacuolar vesicles, which could act as internal stores or recycling vesicles to provide the metal cofactor to key copper-dependent processes such as photosynthesis.     

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA_f^Met). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA_f^Met, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA_f^Met to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA_f^Met. This accessory factor might be unnecessary when formylated Met-tRNA_f^Met is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA_f^Met due to loss of the MIS1 gene that encodes the mitochondrial C₁-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.     

Two ubiquitin-associated ER proteins interact with COPT copper transporters and modulate their accumulation

The endoplasmic reticulum (ER) contains an elaborate protein quality control network that promotes protein folding and prevents accumulation of misfolded proteins. Evolutionarily conserved UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2) is involved in ER-associated protein degradation in metazoans. We have previously reported that two close UBAC2 homologs from Arabidopsis (Arabidopsis thaliana) not only participate in selective autophagy of ER components but also interact with plant-specific PATHOGEN-ASSOCIATED MOLECULAR PATTERN (PAMP)-INDUCED COILED COIL (PICC) protein to increase the accumulation of POWDERY MILDEW-RESISTANT 4 callose synthase. Here, we report that UBAC2s also interacted with COPPER (Cu) TRANSPORTER 1 (COPT1) and plasma membrane-targeted members of the Cu transporter family. The ubac2 mutants were significantly reduced in both the accumulation of COPT proteins and Cu content, and also displayed increased sensitivity to a Cu chelator. Therefore, UBAC2s positively regulate the accumulation of COPT transporters, thereby increasing Cu uptake by plant cells. Unlike with POWDERY MILDEW RESISTANCE 4, however, the positive role of UBAC2s in the accumulation of COPT1 is not dependent on PICC or the UBA domain of UBAC2s. When COPT1 was overexpressed under the CaMV 35S promoter, the increased accumulation of COPT1 was strongly UBAC2-dependent, particularly when a signal peptide was added to the N-terminus of COPT1. Further analysis using inhibitors of protein synthesis and degradation strongly suggested that UBAC2s stabilize newly synthesized COPT proteins against degradation by the proteasome system. These results indicate that plant UBAC2s are multifunctional proteins that regulate the degradation and accumulation of specific ER-synthesized proteins.

Two endoplasmic reticulum-resident UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2s play an important role in copper accumulation through interacting COPT copper transporters and modulating their accumulation.     

Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex

Background

In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface.

Methodology/Principal Findings

We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4⁺ and ctr5⁺ genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc₁₂ displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag₂ failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane.

Significance

These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability.     

Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNA^Arg-tRNA^Asp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNA_i^Met precursors and show that precursor tRNA_i^Met accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA_i^Met, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg²⁺-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.     

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.     

Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12

We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu²⁺ inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met³⁸² of TM3 are involved in a Cu²⁺-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.     

A Highlights from MBoC Selection: Ang2/Fat-Free Is a Conserved Subunit of the Golgi-associated Retrograde Protein Complex

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.     

Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNA_i^Met. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNA_i^Met turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNA_i^Met turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.     

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.     

Calcium‐ and potassium‐permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production

Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH^?), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper‐transport activity: Col‐0, high‐affinity copper transporter COPT1‐overexpressing (C1^OE) seedlings, and T‐DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm ) stimulated a dose‐dependent Gd³⁺ and verapamil sensitive net Ca²⁺ influx in the root apex but not in mature zone. C1^OE also showed a fivefold higher Cu‐induced K⁺ efflux at the root tip level compared with Col‐0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1^OE seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd³⁺. Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH^?‐sensitive Ca²⁺ and K⁺ transport systems.