Dual effect of tamoxifen on arterial KCa channels does not depend on the presence of the beta1 subunit

Tamoxifen has been reported to directly activate large conductance calcium-activated potassium (KCa) channels through the KCa beta1 subunit, suggesting a cardio-protective role of this compound. The present study using knock-out (KO) mice for the KCa channel beta1 subunit was aimed at understanding the molecular mechanisms of the effects of tamoxifen on arterial smooth muscle KCa channels. Single channel studies were conducted in excised patches from cerebral artery myocytes from both wild-type and KO animals. The present data demonstrated that tamoxifen can inhibit arterial KCa channels due to a major decrease in channel open probability (P(o)), a mechanism different from the reduction in single channel amplitude reported previously and also observed in the present work. A tamoxifen-induced decrease in P(o) was present in arterial KCa channels from both wild-type and beta1 KO animals. This inhibition was concentration-dependent and partially reversible with a half-maximal concentration constant IC(50) of 2.6 microm. The effect of tamoxifen was actually dual Single channel kinetic analysis showed that tamoxifen shortens both mean closed time and mean open time; the latter is probably due to an intermediate duration voltage-independent blocking mechanism. Thus, tamoxifen block would predominate when KCa channel P(o) is >0.1-0.2, limiting the maximum P(o), whereas a leftward shift in voltage or Ca(2+) activation curves can be observed for P(o) values lower than those values. This dual effect of tamoxifen appears to be independent of the beta1 subunit. The molecular specificity of tamoxifen, or eventually other xenoestrogen derivatives, for the KCa channel beta1 subunit is uncertain.     

Interaction of agitoxin2, charybdotoxin,and iberiotoxin with potassium channels: selectivity between voltage-gated and Maxi-K channels

To gain insight into the molecular determinants that define the specificity of interaction of pore-blocking peptides, such as agitoxin 2 (AgTX2), charybdotoxin (ChTX), and iberiotoxin (IbTX) with the Shaker-type voltage-gated potassium channel Kv1.3, or the large-conductance Ca(2+)-activated K(+) (Maxi-K) channel, homology models of these channels were generated based on the crystal structure of the bacterial, KcsA, potassium channel. Peptide-channel complexes were analyzed to evaluate the predicted interaction interfaces between the peptides and the channels' outer vestibules. The docking model, for either AgTX2 or ChTX with the Kv1.3 channel, predicts a novel hydrogen bonding interaction between the Asn30 side-chain of the peptide and the Asp381 side-chain of the channel. This interaction is consistent with the >500-fold decreased potency of both AgTX2 and ChTX mutants at position 30 for the Shaker channel [(Ranganathan et al., Neuron 1996;16:131-139); (Goldstein et al., Neuron 1994;12:1377-1388)]. This hydrogen bonding interaction also suggests that Gly30 in IbTX may be the critical determinant for its lack of activity against Shaker Kv channels. The model of the Maxi-K channel reveals a narrower and more structurally restrained outer vestibule in which the aromatic residues Phe266 and Tyr294 may stabilize binding of IbTX and ChTX by pi-pi stacking with the aromatic residues Trp14 and Tyr36 of the peptides. This study also suggests that the extra net negative charge of IbTX is not related to the selectivity of this peptide for the Maxi-K channel.     

Interaction of NUB1 with the proteasome subunit S5a

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.     

An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating

Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of beta1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca(2+) binding) due to the very low open probability in the presence of beta1. In this study, we used a mutation of the alpha subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the beta1 subunit. Effects of beta1 on steady-state open probabilities of both wild-type alpha and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse beta1 has two major effects on channel's gating energetics. beta1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca(2+). Further, P(O) measurements at limiting slope allow us to infer that beta1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca(2+) concentrations. Using the F315Y alpha subunit with deletion mutants of beta1, we also demonstrate that the small N- and C-terminal intracellular domains of beta1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that beta1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.     

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.     

A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alphabeta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alphabeta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alphabeta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alphabeta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting 'competitive' antagonist) binds to this residue.     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

(Xeno)estrogen sensitivity of smooth muscle BK channels conferred by the regulatory beta1 subunit: a study of beta1 knockout mice

Estrogen and xenoestrogens (i.e. agents that are not steroids but possess estrogenic activity) increase the open probability (P(o)) of large conductance Ca(2+)-activated K(+) (BK) channels in smooth muscle. The mechanism of action may involve the regulatory beta1 subunit. We used beta1 subunit knockout (beta1-/-) mice to test the hypothesis that the regulatory beta1 subunit is essential for the activation of BK channels by tamoxifen, 4-OH tamoxifen (a major biologically active metabolite), and 17beta-estradiol in native myocytes. Patch clamp recordings demonstrate BK channels from beta1-/- mice were similar to wild type with the exception of markedly reduced Ca(2+)/voltage sensitivity and faster activation kinetics. In wild type myocytes, (xeno)estrogens increased NP(o) (P(o) x the number of channels, N), shifted the voltage of half-activation (V(12)) to more negative potentials, and decreased unitary conductance. These effects were non-genomic and direct, because they were rapid, reversible, and observed in cell-free patches. None of the (xeno)estrogens increased the NP(o) of BK channels from beta1-/- mice, but all three agents decreased single channel conductance. Thus, (xeno)estrogens increase BK NP(o) through a mechanism involving the beta1 subunit. The decrease in conductance did not require the beta1 subunit and probably reflects an interaction with the pore-forming alpha subunit. We demonstrate regulation of smooth muscle BK channels by physiological (steroid hormones) and pharmacological (chemotherapeutic) agents and reveal the critical role of the beta1 subunit in these responses in native myocytes.     

A TM2 residue in the beta1 subunit determines spontaneous opening of homomeric and heteromeric gamma-aminobutyric acid-gated ion channels

Gamma-aminobutyric acid type A (GABAA) receptors are major inhibitory neurotransmitter-gated ion channels in the central nervous system. GABAA receptors consist of multiple subunits and exhibit distinct pharmacological and channel properties. Of all GABAA receptor subunits, the beta subunit is thought to be a key component for the functionality of the receptors. Certain types of GABAA receptors have been found to be constitutively active. However, the molecular basis for spontaneous opening of channels of these receptors is not totally understood. In this study, we showed that channels that contain the beta1 but not beta3 subunits opened spontaneously when these subunits were expressed homomerically or co-expressed with other types of GABAA receptor subunits in Xenopus oocytes. Using subunit chimeras and site-directed mutagenesis, we localized a key amino acid residue, a serine at position 265, that is critical in conferring an open state of the beta1 subunit-containing GABAA receptors in the absence of agonist. Moreover, some point mutations of Ser-265 also produced constitutively active channels. The magnitude of spontaneous activity of these receptors was correlated with the molecular volume of the residue at 265 for both homomeric and heteromeric GABAA receptors, suggesting that the spontaneous activity of the beta1 subunit-containing GABAA receptors may be mediated through a similar molecular mechanism that is dependent on the molecular volume of the residue at 265.     

Interaction of the Bacillus subtilis RNase P with the 30S ribosomal subunit

Ribonuclease P (RNase P) is a ribozyme required for the 5' maturation of all tRNA. RNase P and the ribosome are the only known ribozymes conserved in all organisms. We set out to determine whether this ribonucleoprotein enzyme interacts with other cellular components, which may imply other functions for this conserved ribozyme. Incubation of the Bacillus subtilis RNase P holoenzyme with fractionated B. subtilis cellular extracts and purified ribosomal subunits results in the formation of a gel-shifted complex with the 30S ribosomal subunit at a binding affinity of approximately 40 nM in 0.1 M NH(4)Cl and 10 mM MgCl(2). The complex does not form with the RNase P RNA alone and is disrupted by a mRNA mimic polyuridine, but is stable in the presence of high concentrations of mature tRNA. Endogenous RNase P can also be detected in the 30S ribosomal fraction. Cleavage of a pre-tRNA substrate by the RNase P holoenzyme remains the same in the presence of the 30S ribosome, but the cleavage of an artificial non-tRNA substrate is inhibited eightfold. Hydroxyl radical protection and chemical modification identify several protected residues located in a highly conserved region in the RNase P RNA. A single mutation within this region significantly reduces binding, providing strong support on the specificity of the RNase P-30S ribosome complex. Our results also suggest that the dimeric form of the RNase P is primarily involved in 30S ribosome binding. We discuss several models on a potential function of the RNase P-30S ribosome complex.     

The human heart and rat brain IIA Na+ channels interact with different molecular regions of the beta1 subunit

The alpha subunit of voltage-gated Na(+) channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory beta(1), but not the beta(2) subunit. In the present study, we used beta(1)/beta(2) chimeras to identify molecular regions within the beta(1) subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na(+) channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the beta(1)/beta(2) chimeras with rat brain IIA channels. In agreement with previous studies, the beta(1) extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the beta(1) subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the beta(1) subunit was required. An exchange of the beta(1) membrane anchor by the corresponding beta(2) subunit region almost completely abolished the effects of the beta(1) subunit on hH1, suggesting that the beta(1) membrane anchor plays a crucial role for the modulation of the cardiac Na(+) channel isoform. It is concluded that the beta(1) subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.     

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

Summary The Host Factor required for in vitro coliphage Q RNA replication, a heat-stable RNA binding protein present in uninfectedEscherichia coli, has been detected by both immunological and functional tests inAcinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa andPseudomonas putida. It was not detectable by these criteria inBacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata orSaccharomyces cerevisiae. InEscherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demostrated here that this association is specific for the 30S ribosomal subunit.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding

The oligomerization and aggregation of the amyloid-β (Aβ) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of Aβ-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying Aβ interactions is currently very limited. We have earlier determined the structure of Aβ(1-40) in complex with the affibody protein Z_Aβ3, a selected binding protein based on a three-helix bundle scaffold (Z domain). Z_Aβ3 is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z_Aβ3 binds to the central and C-terminal part of Aβ (residues 17-36), which adopts a β-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z_Aβ3:Aβ(1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z_Aβ3 is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z_Aβ3:Aβ(1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both Aβ and Z_Aβ3 fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and Aβ is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of Aβ. Options for improvement of Z_Aβ binding proteins are discussed.     

Interaction of the FAFB-containing subunit with the Photosystem 1 core heterodimer

The structure of the predicted amino acid sequence in the F_X domain of Photosystem 1 was studied by molecular modeling and a working hypothesis was developed for the functional interaction of PsaC with the core heterodimer. We propose that the intervening sequences between homologous cysteines in the F_X cluster form two flexible loops and participate in the binding of PsaC, and that the arginine residues in the two surface-exposed loops may promote the interaction between the P₇₀₀–F_X core and the subunit. The model was tested experimentally; chemical modification of arginine residues in the P₇₀₀–F_X core using phenylglyoxal prevented reconstitution of the core with PsaC and PsaD after insertion of FeS clusters in vitro. Treatment of the P₇₀₀–F_X core with trypsin also prevented reconstitution of terminal electron transfer to F_AF_B, although neither treatments affected the electron transfer to F_X as judged by flash kinetic spectrophotometry. Electron transfer in the P₇₀₀–F_AF_B complex was not impaired by either phenylglyoxal or trypsin treatment indicating that the small subunit(s) protect the arginine residues that become chemically modified or cleaved. The data are consistent with the working model and point to additional experiments designed to identify the specific residues involved in the interaction between the P₇₀₀–F_X core and PsaC.Abbreviations PG- phenylglyoxal - PS 1- Photosystem 1     

Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.     

Interaction of translation initiation factor 3 with the 30S ribosomal subunit.

Hydroxyl radical footprinting and directed probing from Fe(II)-derivatized IF3 have been used to map the interaction of IF3 relative to 16S rRNA and tRNA(Met)(f) in the 30S ribosomal subunit. Our results place the two domains of IF3 on opposite sides of the initiator tRNA, with the C domain at the platform interface and the N domain at the E site. The C domain coincides with the location of helix 69 of 23S rRNA, explaining the ability of IF3 to block subunit association. The N domain neighbors proteins S7 and S11 and may interfere with E site tRNA binding. Our model suggests that IF3 influences initiator tRNA selection indirectly.     

Mechanism of action of the wheat germ ribosome dissociation factor: Interaction with the 60 S subunit

Recently a ribosome dissociation factor that stimulates natural mRNA translation has been isolated from extracts of wheat germ. In this investigation, we have studied the subunit site of action of the purified ribosome dissociation factor (eucaryotic initiation), eIF-6. The following evidence strongly indicates that eIF-6 acts as a dissociation factor by binding to the 60 S ribosomal subunit and preventing its interaction with the 40 S subunit. Incubation of 60 S subunits with eIF-6 reduces the formation of 80 S monosomes when 40 S subunits are subsequently added at 5 mm Mg²⁺. The 40 S subunits preincubated with eIF-6 reassociate normally with 60 S subunits. ¹⁴C-labeled eIF-6 binds to 60 S subunits but not to 40 S subunits. Slight binding to 80 S ribosomes is also observed. The interaction of eIF-6 with the 60 S subunit requires an elevated temperature, and occurs rapidly at 37 °C.     

Gating and conductance properties of BK channels are modulated by the S9-S10 tail domain of the alpha subunit. A study of mSlo1 and mSlo3 wild-type and chimeric channels.

The COOH-terminal S9-S10 tail domain of large conductance Ca(2+)-activated K(+) (BK) channels is a major determinant of Ca(2+) sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416-421). To investigate whether the tail domain also modulates Ca(2+)-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:3509-3516). Compared with mSlo1 channels, mSlo3 channels showed little Ca(2+) sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca(2+); reduced the Ca(2+) sensitivity greatly by allowing only partial activation by Ca(2+) and by reducing the Hill coefficient for Ca(2+) activation; decreased the voltage dependence approximately 28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance approximately 14%; decreased the K(d) for block by TEA(i) approximately 30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca(2+) sensitivity.