Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Guanosine 5', alpha-beta-methylene, triphosphate, a novel GTP analog, causes persistent activation of adenylate cyclase: evidence against pyrophosphorylation mechanism.

To test the hypothesis that guanine nucleotides activate adenylate cyclase by a covalent mechanism involving pyrophosphorylation of the enzyme, we studied the effect of a novel GTP analog, guanosine 5′, α-β-methylene triphosphate (Gp(CH₂)pp), with a methylene bond in the α-β-position that is stable to enzymatic hydrolysis. Gp(CH₂)pp was as effective as GTP in stimulating rat reticulocyte adenylate cyclase in the presence of isoproterenol. Previously only guanine nucleotides with modified terminal phosphates such as guanylyl 5′-imidodiphosphate (Gpp(NH)p) were thought capable of causing persistent activation of adenylate cyclase. Gp(CH₂)pp, however, caused persistent activation of rat reticulocyte and turkey erythrocyte adenylate cyclase. We conclude that guanine nucleotides do not activate adenylate cyclase by a pyrophosphorylation mechanism and that a modified γ-phosphate is not essential in guanine nucleotides for generation of the irreversibly-activated enzyme state.     

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer. Here we report the assembly properties of ClpA in the presence of the nucleoside diphosphates and triphosphates ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-(β,γ-imido)triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and adenosine diphosphate beryllium fluoride. In addition to examining the assembly of ClpA in the presence of various nucleotides and nucleotide analogues, we have also correlated the assembly state of ClpA in the presence of these nucleotides with both polypeptide binding activity and enzymatic activity, specifically ClpA-catalyzed polypeptide translocation. Here we show that all of the selected nucleotides, including ADP, promote the assembly of ClpA. However, only adenosine 5′-[γ-thio]triphosphate and adenosine 5′-(β,γ-imido)triphosphate promote the formation of an oligomer of ClpA that is active in polypeptide binding and translocation. These results suggest that the presence of γ phosphate may serve to switch ClpA into a conformational state with high peptide binding activity, whereas affinity is severely attenuated when ADP is bound.     

Dynamic affinity chromatography of heavy meromyosin subfragment-1

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg²⁺ or Ca²⁺. Splitting of bound ATP was followed by using [γ-^{3 2}P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the P_i-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Purification and properties of galactokinase from Tetrahymena thermophila

Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The K_m values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg²⁺ >Co²⁺ >Mn²⁺ >Fe²⁺. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.     

Nucleotide-Induced Flexibility Change in Neck Linkers of Dimeric Kinesin as Detected by Distance Measurements Using Spin-Labeling EPR

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼ 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered.We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.     

Di- and triphosphate derivatives of acyclo- and arabinosylguanine effects on the polymerization of purified tubulin

Glutamate- and nucleotide-dependent polymerization of purified calf brain tubulin was used as a model system to study interactions of ribose-modified GDP and GTP analogs with tubulin. Earlier studies (Hamel, E., and Lin, C.M. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3368–3372) were extended to three additional sets of analogs: the di- and triphosphate derivatives of 9-β-D-arabinofuranosylguanine (araGDP and araGTP) and acycloguanosine (9-(2-hydroxyethoxymethyl)guanine) (acycloGDP and acycloGTP), as well as the periodate-oxidized and borohydride-reduced derivatives of GDP and GTP (ox-redGDP and ox-redGTP). Disruption of the ribose ring in ox-redGTP resulted in major loss of activity relative to GTP in supporting tubulin polymerization, although the analog's deficiency may result from an inability to displace GDP from the exchangeable site rather than a direct effect on the polymerization reaction itself. The poor activity of ox-redGTP could be largely reversed if nucleoside diphosphate kinase was added to the reaction mixture. Removal of the 2′ and 3′ carbons entirely, in the form of acycloGTP, resulted in only minimal loss of activity relative to GTP. AraGTP, on the other hand, was more active than GTP in supporting tubulin polymerization. All three GDP analogs were much less effective than GDP in inhibiting tubulin polymerization, although araGDP was significantly more inhibitory than acycloGDP or ox-redGDP. Relative inhibitory activity of these and additional GDP analogs was the same whether GTP or a GTP analog was used to support tubulin polymerization.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.

2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.

3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.

4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-³²P_i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.

5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.

6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.

7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD⁺ by succinate.

8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans I. Inhibition by heparin of gonadotropin- stimulated ovarian adenylate cyclase

Heparin inhibits (I₅₀ = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.     

Structures of the Ca-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study

We studied binding of ATP and of the ATP analogs adenosine 5′-(β,γ-methylene)triphosphate (AMPCP) and β,γ-imidoadenosine 5′-triphosphate (AMPPNP) to the Ca²⁺-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca₂E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca²⁺ concentration (10 mM). They are likely due to a different position of the γ-phosphate which affects the β-sheet in the P domain.     

Active Ca2+ transport by membrane vesicles from pigeon erythrocytes Stimulation by amino acids,ATP, GTP,P1 and some other cell constituents

Pretreatment of pigeon erythrocyte membrane vesicles with amino acids, ATP, GTP, P_i and some other simple cell constituents (singly and in combination) causes an increase in ATP-dependent Ca²⁺-uptake activity of vesicles upon subsequent incubation with ⁴⁵Ca²⁺ after removal of the above agents from the ‘i’ face. Amino acids augment the stimulation by all stimulatory agents and are required for stimulation by P_i. The effects of amino acids, ATP, GTP and P_i all occur at physiological concentrations. Many if not all of the effects of the mixture of amino acids that occur naturally in the cells can be accounted for by the group transported by the ‘ASC’ transport system of Christensen (Christensen H.N. (1975) Biological Transport, 2nd edn., W.A. Benjamin, Inc., Reading, MA), but not by any single amino acid at its physiological concentration. The effects of ATP and GTP are not mimicked by their non-hydrolysable β, γ-imido analogues nor by the corresponding 3′, 5′-cyclic monophosphates. None of the effects described appears to involve calmodulin. We suggest that amino acid transport plays a role in metabolic regulation through effects on cell [Ca²⁺]. Analogous effects on cell [Ca²⁺] may be involved in the action of the many hormones which augment amino acid accumulation by the ‘A’ amino acid transport system.     

Extracellular Nucleotide Catabolism in Aortoiliac Bifurcation of Atherosclerotic ApoE/LDLr Double Knock Out Mice

Atherosclerosis is a consequence of diverse pathologies that could be affected by signaling mediated by nucleotides and their metabolites. Concentration of specific nucleotide derivatives in the proximity of purinergic receptors is controlled by extracellular enzymes such as ecto-nucleoside triphopsphate diphosphohydrolase (eNTPD), ecto-5′-nucleotidase (e5NT), and ecto-adenosine deaminase (eADA). To estimate changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall, aortoiliac bifurcation of ApoE/LDLr (–/–) mice was perfused with solution containing adenosine-5′-triphosphate (ATP), adenosine-5′-monophosphate (AMP) or adenosine. Formation of the product of eNTPD, e5NT or eADA was measured by high performance liquid chromatography (HPLC). The most significant difference between ApoE/LDLr (–/–) and wild-type mice was several times higher rate of conversion of adenosine to inosine catalyzed by eADA activity. This highlights potential decrease in intravascular adenosine concentration in atherosclerosis.     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.