Inhibition of [3H]batrachotoxinin A-20alpha-benzoate binding to sodium channels and sodium channel function by endocannabinoids

A number of putative endocannabinoids were found to modify the binding of [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) to site 2 on voltage-gated sodium channels of mouse brain and achieve functional inhibition of sodium channels in vitro. 2-Arachidonoyl-glycerol (2-AG), arachidonoyl glycerol ether (AGE), N-arachidonoyl-dopamine (NADA) gave almost complete inhibition of [(3)H]BTX-B binding with IC(50) values of 90.4, 51.2 and 20.7 microM, respectively. The CB1 receptor antagonist AM251 (2 microM) had no effect on the displacement of radioligand by these endocanabinoids. Arachidonoyl-glycine (A-Gly) and arachidonoyl-GABA (A-GABA) were apparently less effective inhibitors of [(3)H]BTX-B binding giving 14.8+/-2.2 and 23.9+/-4.8% inhibition at 100 microM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 microM A-GABA was increased by 60.3+/-6.3% (P<0.05). Scatchard analyses showed that 2-AG, AGE and NADA reduce the binding of [(3)H]BTX-B by lowering B(max) although increases in K(D) were also evident for AGE and NADA. Our kinetic experiments found that 2-AG, AGE and NADA increase the dissociation velocity of radioligand from site 2 on sodium channels demonstrating that these endocannabinoids operate as allosteric inhibitors of [(3)H]BTX-B binding. 2-AG, AGE and NADA inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes at low micromolar concentrations and again the capacities of A-Gly and A-GABA to inhibit this response were less pronounced. The three most effective endocannabinoids (2-AG, AGE and NADA) were then examined in a synaptosomal transmitter release assay where they were observed to inhibit sodium channel- (veratridine-dependent) release of l-glutamate and GABA in the low micromolar range. These effects also occurred through a mechanism that was not influenced by 2 microM AM251. It is concluded that direct inhibition of sodium channel function leading to reduced neuronal excitation and depression of presynaptic release of amino acid transmitters is a property shared by several endocannabinoids.     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Specific binding of a calcium channel activator, [3H]BAY k 8644, to membranes from cardiac muscle and brain

BAY k 8644 is a member of a new class of drugs that directly activates Ca2+ channels. This 1,4-dihydropyridine was found to bind to both high and low affinity sites on rabbit ventricular microsomes and guinea pig brain synaptosomes. The dissociation constant obtained from Scatchard analysis with [3H]BAY k 8644 was 2 to 3 nM for the high affinity binding site, and the estimated maximal number of binding sites was 0.8 and 0.4 pmol/mg protein for heart and brain membranes, respectively, at 15 degrees C. Competition between nitrendipine and [3H]BAY k 8644 indicated a common high affinity binding site for Ca2+ channel activators and antagonists. The results suggest that the 1,4-dihydropyridine Ca2+ channel antagonists do not act as simple channel plugs.     

3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [3H]PN200-110, and the alkaloid, [3H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.     

Specific high affinity binding of the calcium channel antagonist, [3H]nitrendipine, to rat liver microsomes

Some key properties of the binding of [3H]nitrendipine, an analogue of the 1,4-dihydropyridine, nifedipine, to a plasma membrane enriched microsomal fraction from the rat liver are described. Specific binding was saturable, linear with protein concentration, and reversible. The apparent equilibrium dissociation constant, KD, was 4.20 +/- 0.22 nM and the maximum density of binding, Bmax, was 3.02 +/- 0.17 pmol/mg of protein determined from Scatchard analysis of binding at 10 degrees C. Inhibition of binding was specific for dihydropyridines with competitive inhibition being noted with nifedipine and 4-chloronifedipine, as well as BAY K-8644, a calcium channel agonist. A biphasic displacement curve was recorded for methoxy verapamil (D-600), and a triphasic competition curve with lanthanum (La3+), and diltiazem demonstrated competitive kinetics. The high affinity binding site for nitrendipine in the liver, although having some similar properties to those sites described in skeletal muscle, would appear to be distinctive with respect to its unique sensitivity to D-600 and diltiazem. We speculate that this binding site may represent a Ca2+ channel responsible for regulating Ca2+ influx and hepatic glycogenolysis.     

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to - 1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.     

Characterization of alpha-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes

Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.     

Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin

The inhibitory effect of leupeptin on [3H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [3H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [3H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).     

Procaine isothiocyanate: An irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels

[³H]Batrachotoxinin-A benzoate ([³H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [³H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative ([³H]PRIT) have been prepared. PRIT inhibits the binding of [³H]BTX-B in a noncompetitive, irreversible manner (apparent K_i=13 M) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (K_i=40 M). The dissociation rate of [³H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [³H]BTX-B is achieved in the presence of 0.98 M PRIT. [³H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.     

Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.     

Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure

Pickar, Joel G., John P. Mattson, Steve Lloyd, and TimothyI. Musch. Decreased[³H]ouabainbinding sites in skeletal muscle of rats with chronic heart failure.J. Appl. Physiol. 83(1): 323-329, 1997.Abnormalities intrinsic to skeletal muscle are thought tocontribute to decrements in exercise capacity found in individualswith chronic heart failure (CHF).Na⁺-K⁺-adenosinetriphosphatase(the Na⁺ pump) is essential formaintaining muscle excitability and contractility. Therefore, weinvestigated the possibility that the number and affinity ofNa⁺ pumps in locomotor muscles ofrats with CHF are decreased. Myocardial infarction (MI) was induced in8 rats, and a sham operation was performed in 12 rats. The degree ofCHF was assessed ~180 days after surgery. Soleus and plantarismuscles were harvested, and Na⁺pumps were quantified by using a[³H]ouabain bindingassay. At the time of muscle harvest, MI and sham-operated rats weresimilar in age (458 ± 54 vs. 447 ± 34 days old, respectively).Compared with their sham-operated counterparts, MI rats had asignificant amount of heart failure, right ventricular-to-body weightratio was greater (48%), and the presence of pulmonary congestion wassuggested by an elevated lung-to-body weight ratio (29%). Leftventricular end-diastolic pressure was significantly increased in theMI rats (11 ± 1 mmHg) compared with the sham-operated controls (1 ± 1 mmHg). In addition, mean arterial blood pressure was lower inthe MI rats compared with their control counterparts. [³H]ouabain bindingsites were reduced 18% in soleus muscle (136 ± 12 vs. 175 ± 13 pmol/g wet wt, MI vs. sham, respectively) and 22% in plantaris muscle(119 ± 12 vs. 147 ± 8 pmol/g wet wt, MI vs. sham,respectively). The affinity of these[³H]ouabain bindingsites was similar for the two groups. The relationship between thereduction in Na⁺ pump number andthe reduced exercise capacity in individuals with CHF remains to bedetermined.

     

Specific binding of [3H]phenytoin in the human brain

Competition between cold phenytoin and [3H]phenytoin binding was observed in normal human brain. Binding was observed in all areas examined. The highest number of sites was in the amygdala (a total of 717.71 fmol/mg protein) and the lowest in the Brodman area (BA) 4 of the motor cortex (153.91 fmol/mg protein) and cerebellar cortex (154.4 fmol/mg protein). In three areas, amygdala, cortex area BA 38 (inferior parietal lobe), and cortex area BA 8 (premotor cortex), two sets of binding sites were observed. In these areas the Kd for the higher affinity sites ranged from 35 to 116 nM, and for the lower affinity site, from 328 to 866 nM. In the four areas where only one binding site was observed the KdS ranged from 164 to 311 nM and the Scatchard plot was linear.     

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.     

High affinity inhibition of [3H]-flunitrazepam binding to brain benzodiazepine receptors by CGS 9896, a novel pyrazoloquinoline

We had previously reported the extensive purification of snRNPs (ribonucleoprotein complexes containing small nuclear RNAs) from HeLa hnRNP (ribonucleoprotein complexes containing the heterogenous nuclear RNA) by taking advantage of their ability to withstand centrifugation in cesium chloride containing 0.5% sarkosyl (C. BRUNEL et al 1981, Nucleic Acids Research, 9, 815–830). Such purified snRNPs contained the five nucleoplasmic snRNAs (U₁, U₂, U₄, U₅ and U₆) and a very simple protein complement of 4–5 polypeptides between 10 and 14,000 daltons. The possible involvement of U₁ in messenger RNA splicing makes it crucial to obtain in pure form the individual U₁ snRNP, if an in vitro splicing system is to be reconstructed. The present work reports the purification of U₁ snRNP and shows that it contains exactly the same polypeptides as the mixture of all five snRNPs suggesting that the specific function, if any, depends on the RNA moiety.