Mutant Beta Subunits Research Articles

Mitochondrial ATP Synthase beta-Subunit Affects Plastid Retrograde Signaling in Arabidopsis.

Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes (PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase beta-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling in Arabidopsis thaliana. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in the mutants affected in the AT5G08670 gene encoding the mitochondrial ATP synthase beta-subunit, compared to wild-type (WT) seedlings when treated with lincomycin (LIN) or norflurazon (NF). Further studies indicated that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in the AT5G08670 mutant seedlings treated with LIN. These changes might be linked to the modulation of some transcription factors (TFs), such as LHY (Late Elongated Hypocotyl), PIF (Phytochrome-Interacting Factors), MYB, WRKY, and AP2/ERF (Ethylene Responsive Factors). These findings suggest that the activity of mitochondrial ATP synthase significantly influences plastid retrograde signaling.

Insights Into the Differential Desensitization of α4β2 Nicotinic Acetylcholine Receptor Isoforms Obtained With Positive Allosteric Modulation of Mutant Receptors.

The development of highly efficacious positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChR) has proven useful in defining the ligand dependence of the conformational dynamics of α7 receptors. No such effective modulators are known to exist for the α4β2 nAChR of the brain, limiting our ability to understand the importance of desensitization for the activity profile of specific ligands. In this study, we used mutant β2 subunits that allowed the use of the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) to probe the desensitizing effects of nicotinic ligands on the two forms of α4β2 receptors; high sensitivity (HS) (two α4 and three β2 subunits) and low sensitivity (LS) (three α4 and two β2 subunits). A total of 28 different ligands of 8 different categories, based on activity and selectivity, were tested for their ability to induce TQS-sensitive desensitization of HS and LS α4β2 receptors. Results confirm that HS α4β2 receptor responses are strongly limited by desensitization, by at least an order of magnitude more so than the responses of LS receptors. The activation of α4β2 receptors by the smoking-cessation drugs cytisine and varenicline is strongly limited by desensitization, as is the activation of LS receptors by the HS-selective agonists 6-[5-[(2S)-2-Azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol dihydrochloride and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate. The evaluation of drugs previously identified as α7-selective agonists revealed varying patterns of α4β2 cross-desensitization that were predictive of the effects of these drugs on the activation of wild-type α4β2 receptors by acetylcholine, supporting the utility of TQS-sensitive receptors for the development of focused therapeutics. SIGNIFICANCE STATEMENT: To varying degrees, ligands regulate the balance of active and desensitized states of the two forms of the primary nAChR subtypes in brain. Using mutant beta subunits, an allosteric modulator can reverse ligand-induced desensitization, revealing the differential desensitization of the receptors by specific ligands. This study shows that drugs believed to be selective for therapeutic targets may cross-desensitize other targets and that, within a class of drugs, improved specificity can be achieved by using agents that reduce such cross-desensitization.

Development of transgenic rice containing a mutated β subunit of soybean β-conglycinin for enhanced phagocytosis-stimulating activity

Improving the nutraceutical value of rice would positively impact the health and well-being of rice consumers worldwide. Based on the three-dimensional structure of soybean beta-conglycinin, we designed a beta subunit with a strong phagocytosis-stimulating activity (mbeta subunit). Here, we describe the genetic modification and production of rice seeds containing the mbeta subunit as part of our aim to develop a food material that promotes human health. The mbeta subunit folded correctly and was accumulated in the protein body II of rice seeds at a level similar to wild-type beta subunit. Mutant beta subunit purified from transgenic rice seeds exhibited high phagocytosis-stimulating activity, demonstrating its potential value in enhancing the nutritional value of rice.

Ankyrin-G promotes cyclic nucleotide-gated channel transport to rod photoreceptor sensory cilia.

Cyclic nucleotide-gated (CNG) channels localize exclusively to the plasma membrane of photosensitive outer segments of rod photoreceptors where they generate the electrical response to light. Here, we report the finding that targeting of CNG channels to the rod outer segment required their interaction with ankyrin-G. Ankyrin-G localized exclusively to rod outer segments, coimmunoprecipitated with the CNG channel, and bound to the C-terminal domain of the channel beta1 subunit. Ankyrin-G depletion in neonatal mouse retinas markedly reduced CNG channel expression. Transgenic expression of CNG channel beta-subunit mutants in Xenopus rods showed that ankyrin-G binding was necessary and sufficient for targeting of the beta1 subunit to outer segments. Thus, ankyrin-G is required for transport of CNG channels to the plasma membrane of rod outer segments.

Design of Mutant β2Subunits as Decoy Molecules to Reduce the Expression of Functional Ca2+Channels in Cardiac Cells

Calcium influx through long-lasting ("L-type") Ca(2+) channels (Ca(V)) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca(2+) overload, and interventions that reduce expression of the pore-forming alpha(1) subunit may alleviate cytosolic Ca(2+) excess. As a molecular approach to disrupt the assembly of Ca(V)1.2 (alpha(1C)) channels at the cell membrane, we targeted the Ca(2+) channel beta(2) subunit, an intracellular chaperone that interacts with alpha(1C) via its beta interaction domain (BID) to promote Ca(V)1.2 channel expression. Recombinant adenovirus expressing either the full beta(2) subunit (Full-beta(2)) or truncated beta(2) subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-beta(2) at the surface membrane was associated with increased Ca(2+) current mainly attributed to Ca(V)1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the alpha(1C) protein of Ca(V)1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca(2+) current. C-BID and BID also attenuated Ca(2+) current associated with another L-type Ca(2+) channel, Ca(V)1.3, but they did not reduce transient Ca(2+) currents attributed to Ca(V)3 channels. These results suggest that beta(2) subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca(2+) channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca(2+) overload.

F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits

F1-ATPase, the catalytic part of FoF1-ATP synthase, rotates the central gamma subunit within the alpha3beta3 cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at approximately 200 degrees and carries out a third catalytic event at approximately 320 degrees. This demonstrates the concerted nature of the F1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the gamma subunit with a 120 degrees phase difference, a process we describe as a 'sequential three-site mechanism'.

Contribution of the C-terminal region to the thermostability of the archaeal group II chaperonin from Thermococcus sp. strain KS-1

Chaperonin is a double ring-shaped oligomeric protein complex, which captures a protein in the folding intermediate state and assists its folding in an ATP-dependent manner. The chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, is a group II chaperonin and is composed of two distinct subunits, alpha and beta. Although these subunits are highly homologous in sequence, the homo-oligomer of the beta-subunit is more thermostable than that of the alpha-subunit. To identify the region responsible for this difference in thermostability, we constructed domain-exchange mutants. The mutants containing the equatorial domain of the beta-subunit were more resistant to thermal dissociation than the mutants with that of the alpha-subunit. Thermostability of a beta-subunit mutant whose C-terminal 22 residues were replaced with those of the alpha-subunit decreased to the comparable level of that of the alpha-subunit homo-oligomer. These results indicate that the difference in thermostability between alpha- and beta-subunits mainly originates in the C-terminal residues in the equatorial domain, only where they exhibit substantial sequence difference.

Detection of Open and Closed Conformations of Tryptophan Synthase by 15N-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance of Bound 1-15N-L-Tryptophan

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.

Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system.

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.

Stepping Rotation of F1-ATPase with One, Two, or Three Altered Catalytic Sites That Bind ATP Only Slowly

F(1)-ATPase is an ATP hydrolysis-driven motor in which the gamma subunit rotates in the stator cylinder alpha(3)beta(3). To know the coordination of three catalytic beta subunits during catalysis, hybrid F(1)-ATPases, each containing one, two, or three "slow" mutant beta subunits that bind ATP very slowly, were prepared, and the rotations were observed with a single molecule level. Each hybrid made one, two, or three steps per 360 degrees revolution, respectively, at 5 microm ATP where the wild-type enzyme rotated continuously without step under the same observing conditions. The observed dwell times of the steps are explained by the slow binding rate of ATP. Except for the steps, properties of rotation, such as the torque forces exerted during rotary movement, were not significantly changed from those of the wild-type enzyme. Thus, it appears that the presence of the slow beta subunit(s) does not seriously affect other normal beta subunit(s) in the same F(1)-ATPase molecule and that the order of sequential catalytic events is faithfully maintained even when ATP binding to one or two of the catalytic sites is retarded.

Mutational Study on the Roles of Disulfide Bonds in the β-Subunit of Gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.

Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase beta subunit in the energy coupling mechanism of the Na+ pump.

The membrane-bound beta subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the beta subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild-type enzyme and the beta subunit mutants N373D, N373L, S382A, S382D, S382T, R389A, and R389D. In these studies, pH profiles, Na+ binding affinities, Hill coefficients, Vmax values and inhibition by Na+ was determined. A prominent result is the complete lack of oxaloacetate decarboxylase activity of the S382A mutant at Na+ concentrations up to 20 mm and recovery of significant activities at elevated Na+ concentrations (KNa approximately 400 mm at pH 6.0), where the wild-type enzyme is almost completely inhibited. These results indicate impaired Na+ binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na+ concentrations is uncoupled from the vectorial events of Na+ or H+ translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism, which includes Na+ binding to center I contributed by D203 (region IIIa) and N373 (helix VIII) and center II contributed by Y229 (helix IV) and S382 (helix VIII). These centers are exposed to the cytoplasmic surface in the carboxybiotin-bound state of the beta subunit and become exposed to the periplasmic surface after decarboxylation of this compound. During the countertransport of 2 Na+ and 1 H+ Y229 of center II switches between the protonated and deprotonated Na+-bound state.

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding.

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.

Functions and ATP-Binding Responses of the Twelve Histidine Residues in the TF1-ATPase

The C2 proton signals of all (twelve) histidine residues of the TF1 beta subunit in the 1H-NMR spectrum have been identified and assigned by means of pH change experiments and site-directed substitution of histidines by glutamines. pH and ligand titration experiments were carried out for these signals. Furthermore, the ATPase activity of the reconstituted alpha3beta3gamma complex was examined for the twelve mutant beta subunits. Two of three conserved histidines, namely, His-119 and 324, were found to be important for expression of the ATPase activity. The former fixes the N-terminal domain to the central domain. His-324 is involved in the formation of the interface essential for the alpha3beta3gamma complex assembly. The other conserved residue, His-363, showed a very low pK(a), suggesting that it is involved in the tertiary structure formation. On the binding of a nucleotide, only the signals of His-173, 179, 200, and 324 shifted. These histidines are located in the hinge region, and its proximity, of the beta subunit. This observation provided further support for the conformational change of the beta monomer from the open to the closed form on the binding of a nucleotide proposed by us [Yagi et al. (1999) Biophys. J. 77, 2175-2183]. This conformational change should be one of the essential driving forces in the rotation of the alpha3beta3gamma complex.

Structure-Function of the Putative I-domain within the Integrin β2 Subunit

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 microm), which is most likely located within this region of beta(2).

High Incidence of Propionic Acidemia in Greenland Is Due to a Prevalent Mutation, 1540insCCC, in the Gene for the β-Subunit of Propionyl CoA Carboxylase

Propionyl CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of amino acids, odd-chain fatty acids, and other metabolites. PCC consists of two subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. Inherited PCC deficiency due to mutations in either gene results in propionic acidemia (PA), an autosomal recessive disease. Surprisingly, PA is highly prevalent among Inuits in Greenland. We have analyzed reverse transcriptase-PCR products of the beta-subunit mRNA, to characterize the responsible mutation(s). A 3-bp insertion, 1540insCCC, was found in homozygous form in three patients and in compound heterozygous form in one patient. The resulting PCC has no measurable activity, and the mutant beta-subunit appears to be very unstable. To test the hypothesis that a common mutation is responsible for PA in the Greenlandic Inuit population, 310 anonymous DNA samples of Inuit origin were screened for 1540insCCC. We found a carrier frequency of 5%, which is very high compared with those of most other autosomal recessive diseases. Analysis of alleles of a very closely linked marker, D3S2453, revealed a high degree of linkage disequilibrium between one specific allele and 1540insCCC, suggesting that this mutation may be a founder mutation.

Mutations in the β-Subunit Thr159 and Glu184 of the Rhodospirillum rubrumF0F1 ATP Synthase Reveal Differences in Ligands for the Coupled Mg2+- and Decoupled Ca2+-dependent F0F1 Activities

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.

Functional Regulation of L-type Calcium Channels via Protein Kinase A-mediated Phosphorylation of the β2 Subunit

Activation of protein kinase A (PKA) through the beta-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the beta(2) subunit are of functional significance, we coexpressed wild-type (WT) or mutant beta(2) subunits in tsA-201 cells together with an alpha(1C) subunit, alpha(1C)Delta1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated alpha(1C) subunit was similar to the truncated alpha(1C) subunit isolated from cardiac tissue not only in size ( approximately 190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT beta(2) subunit, voltage-activated Ba(2+) currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the beta(2) subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca(2+) currents may be at least partially caused by phosphorylation of the beta(2) subunit at Ser-478 and Ser-479.

Complexes of the α1C and β Subunits Generate the Necessary Signal for Membrane Targeting of Class C L-type Calcium Channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.

Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.

We have applied the substituted-cysteine-accessibility method (SCAM) to the M2 segment and the M1-M2 loop of the acetylcholine (ACh) receptor beta subunit. Each residue from beta P248 to beta D273 was mutated one at a time to Cys, and the mutant beta subunits were expressed together with wild-type alpha, beta, and delta subunits in Xenopus oocytes. For each of the mutants, the ACh-induced current was near wild-type. The accessibility of the substituted Cys was inferred from the irreversible inhibition or potentiation of ACh-induced current by methanethiosulfonate (MTS) derivatives added extracellularly. Inhibition by MTSethylammonium of beta G255C, in the narrow part of the channel, was mainly due to a reduction in the single-channel conductance. Conversely, potentiation by MTSethylammonium of beta V266C, in a wider part of the channel, was mainly due to an increase in channel open-time. Two substituted Cys at the intracellular end of M2 and three at the extracellular end were accessible to MTSethylammonium in the absence of ACh. Three additional Cys in the middle of M2 and three in the M1-M2 loop were accessible in the presence of ACh. In the presence of ACh, the secondary structure of beta M2 is alpha-helical from beta G255 to beta V266 and extended from beta L268 to beta D273. The accessible residues in beta M2 are remarkably hydrophobic, while the accessible residues in the M1-M2 loop are charged. beta M2, like alpha M2, alpha M1, and beta M1, undergoes widespread structural changes concomitant with gating, but the gate itself is close to the intracellular end of the channel. Many aligned residues in the M2 segments of alpha and beta are not identically accessible, indicating that the two subunits contribute differently to the channel lining.