Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Characterization of neuronal zebra finch GABAA receptors: steroid effects

Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize γ-aminobutyric acid (GABA) type A receptors (GABA_A) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation by 17β-estradiol(E₂), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked currents were inhibited by picrotoxin (10 μmol l⁻¹) and bicuculline methiodide (10 μmol l⁻¹) and potentiated by pentobarbital (100 μmol l⁻¹) and propofol (3 μmol l⁻¹). Loreclezole (10 μmol l⁻¹) potentiated GABA-evoked currents, suggesting the presence of β₂, β₃ and/or β₄ subunits. Diazepam (1 μmol l⁻¹) potentiated currents, while Zn²⁺ (1 μmol l⁻¹) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l⁻¹) potentiated currents, whereas E₂ (1 μmol l⁻¹), 5α- and 5β-DHT (1 μmol l⁻¹), and corticosterone (10 μmol l⁻¹) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABA_A receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little or no acute modulation by sex steroids or corticosterone. Accepted: 12 November 1997     

Tyrosine Kinase Phosphorylation of GABAA Receptor α1, β2 and γ2 Subunits Following Chronic Intermittent Ethanol (CIE) Exposure of Cultured Cortical Neurons of Mice

There is evidence that many of the GABA_A receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol’s regulation of GABA_A receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the β₂- and γ₂- subunits and no effect on α₁-subunit of the GABA_A receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA_A receptor subunits (α₁, β₂, and γ₂) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA_A receptor β₂- and γ₂-subunits following CIE exposure, and no effect on α₁-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA_A receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA_A receptors in a long lasting manner.     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12′ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12′ lysines in the α, γ and δ subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK _a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in β or ε subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK _a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12′ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of α subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel.     

Glycinergic transmission

Inhibition in the mature central nervous system is mediated by activation of γ-aminobutyric acid (GABA_A) and glycine receptors. Both receptors belong to the same superfamily of ligand-gated ion channels and share common transmembrane topology and structural and functional features. Glycine receptors are pentameric ligand-gated anion channels composed of two different subunits, named α und β, that assemble with a fixed stoichiometric ratio of two α to three β subunits. Four genes encoding the α subunits exist, whereas only one gene encoding the β subunit has been detected. Ligand binding occurs at the interface of α and β subunits. The β subunit, which is unable to form homo-oligomeric receptors, is responsible for assembly and channel properties. Moreover, this subunit carries a binding motif for the cytoplasmic protein gephyrin, which is believed to mediate synaptic clustering and anchoring at inhibitory synapses by interacting with the subsynaptic cytoskeleton. Synaptic gephyrin appears to restrict the mobility of glycine receptors diffusing in the plane of the plasma membrane, thereby generating dynamic plasma membrane domains contributing to the plasticity of inhibitory synapses. Glycine receptors are well established as playing important roles in controlling motor functions and sensory signaling in vision and audition and those in the dorsal horn of the spinal cord are now considered to be new targets for pain therapies. Like GABA_A receptors, glycine receptors have been shown to be depolarizing during development. The functional meaning of the developmental switch from excitatory to inhibitory glycine receptor action remains to be elucidated.     

Acetylcholine Receptor Gating is Influenced by the Polarity of Amino Acids at Position 9′ in the M2 Domain

Ligand-gated ion channels contain a conserved leucine at position 9′ (L9′) in the M2 transmembrane domain. We used multiple substitutions at this position in the γ subunit of the mouse acetylcholine receptor (AChR) (γL9′) to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC₅₀) and the channel closing rate constant, α, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9′ position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family. Received: 17 September 1999/Revised: 15 December 1999     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Effect of Chronic Administration of Ethanol on the Regulation of Tyrosine Kinase Phosphorylation of the GABA<Subscript>A</Subscript> Receptor Subunits in the Rat Brain

One of the many pharmacological targets of ethanol is the GABA inhibitory system, and chronic ethanol (CE) is known to alter the polypeptide levels of the GABA_A receptor subunits in rat brain regions. In the present study, we investigated the regulation of the tyrosine kinase phosphorylation of the GABA_A receptor α₁-, β₂- and γ₂-subunits in the rat cerebellum, cerebral cortex and hippocampus following chronic administration of ethanol to the rats. We observed either down-regulation or no change in the tyrosine kinase phosphorylation of the α₁ subunit, whereas there was an up-regulation or no change in the case of β₂- and γ₂-subunits of the GABA_A receptors depending on the brain region following chronic administration of ethanol to the rats. These changes reverted back to the control level following 48 h of ethanol-withdrawal. These results suggest that tyrosine kinase phosphorylation of GABA_A receptors may play a significant role in ethanol dependence.     

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α₂ββ′, α′₂ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.     

Characterization and comparative pharmacological studies of a functional γ-aminobutyric acid (GABA) receptor cloned from the tobacco budworm,Heliothis virescens (Noctuidae:Lepidoptera)

This paper reports the functional expression and pharmacological characterization of a full length complementary deoxyribonucleic acid (cDNA) (pIVY12) cloned from aHeliothis virescens fertilized egg cDNA library that encodes for a γ-aminobutyric acid (GABA) receptor subunit (HVRDL-Ser 285). Two electrode voltage clamp recordings ofXenopus oocytes expressing the HVRDL GABA-gated chloride channel revealed robust chloride ion conductance in response to GABA and the GABA_A receptor agonist, muscimol. Baclofen, a GABA_B agonist had no effect. Phenobarbital showed a positive dose-dependent allosteric modulatory effect, whereas the benzodiazepine, flunitrazepam, had no effect. Chloride conductance was depressed by the novel insecticide, fipronil ((±)-5-amino-1-(2,6 dichloro-α, α, α-trifluoro-p-tolyl)-4-trifluoromethyl-sulfinylpyrazole-3-carbonitrile) and the GABA_A antagonist, picrotoxinin. The HVRDL GABA receptor was insensitive to blockage by dieldrin and the GABA_A antagonist, bicuculline. The comparative actions of fipronil, picrotoxinin and dieldrin were examined on oocytes expressing theH. virescens wild-type (HVRDL-Ser 285), the site-directed mutant (HVRDL-Ala 285), theDrosophila melanogaster Rdl wild-type (DMRDL-Ala 302) and theRdl dieldrin resistant (DMRDL-Ser 302) homo-oligomeric GABA receptors. HVRDL-Ala 285 was 15-fold more sensitive to blockage by fipronil than HVRDL-Ser 285. DMRDL-Ala 302 and DMRDL-Ser-302 showed a similar level of sensitivity to blockage by fipronil. HVRDL-Ser 285 and DMRDL-Ser 302 exhibited a similar level of insensitivity to picrotoxinin. HVRDL-Ala 285 and DMRDL-Ala 302 showed a similar range of picrotoxinin sensitivity. DMRDL-Ala 302 and HVRDL-Ala 285 showed some sensitivity to blockage by dieldrin. Fipronil sensitivity was significantly altered by the serine to alanine mutation at position 285 in the M2 region of the HVRDL subunit, whereas no difference was observed between the DMRDL-Ser 302 and DMRDL-Ala 302 receptors.     

Identification of Major Phylogenetic Branches of Inhibitory Ligand-Gated Channel Receptors

The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC) and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABA_A, Gly, and GluCl receptors, conduct Cl⁻ when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABA_A receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB for non–benzodiazepine binding, and consisting of GABA_A receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits; and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB, composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch IV is called UD for being undefined in specificity. The existence of primitive forms of GABA_A receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123 from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining the identities of newly discovered subunits on the basis of their protein sequences. Received: 15 April 1997 / Accepted: 11 March 1998     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

Modulation of Recombinant GABA Receptor/Channel Subunits by Domain-specific Antibodies in Xenopus Oocytes

To study interaction of specific antibodies with the GABA receptor/channel, antisera were raised against the extracellular domains of the GABA_A receptor/channel β₂ subunit, γ₂ subunit and the GABA_C receptor/channel ρ₁ subunit. The specificity of the antibodies was characterized by immunocytochemistry and by Western blotting of transfected FDC-P1 cells expressing recombinant GABA receptor/channel subunits. The effects of the antibodies on whole-cell currents in Xenopus laevis oocytes expressing homomeric recombinant GABA receptor/channel β₂, γ₂, and ρ₁ were studied using two-microelectrode voltage clamp. In the absence of GABA, anti-α₂, anti-γ₂, and anti-ρ₁ antisera elicited whole-cell currents in oocytes expressing β₂, γ₂, and ρ₁ subunits, respectively. The effect of antibody on channel activation was concentration-dependent. The whole-cell currents induced by anti-β₂ and anti-γ₂ were several-fold greater than those induced by application of 100 μm GABA. In Xenopus oocytes expressing recombinant ρ₁ subunits, GABA-induced whole-cell currents were inhibited by the anti-ρ₁ antibody. In contrast, the GABA-induced whole-cell currents were potentiated several-fold by anti-β₂ and anti-γ₂ antibodies in Xenopus oocytes expressing homomeric β₂ and γ₂ subunits. Our studies indicate that antibodies specific to the N-terminal domain of GABA receptor/channel subunits can modulate the neurotransmitter receptor function. Received: 2 February 2001/Revised: 11 April 2001     

126Gln is the residue of human IL-2 binding to IL-2R γ subunit

The 126Gln of human interleukin-2 (IL-2) is a conserved amino acid residue. After substitution of 126Gln with Asp, the binding abilities of this mutant to different composites of IL-2 receptor (R) subunits have been determined. Results show that 126AspIL-2 has higher affinity to IL-2R α β γ complex and normal affinity to IL-2R α β complex, but loses its binding ability to IL-2R β γ complex, demonstrating that the 126Gln is the residue of human IL-2 which binds to IL-2R γ subunit. Project supported by the “863” Project of China.     

The channel-lining 6′ amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4′-ethynyl-4-<Emphasis Type="Italic">n</Emphasis>-propylbicycloorthobenzoate binding than the 2′ amino acid

The noncompetitive antagonist of ionotropic γ-aminobutyric acid (GABA) receptors 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA_A receptor β3 subunit were stably expressed in S2 cells and examined for their abilities to bind [³H]EBOB to identify the binding site of EBOB. The homo-oligomeric β3 GABA receptor was used as a housefly GABA receptor model, as the β3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1′ position in the M2 region had no effect on [³H]EBOB binding. The A277S mutation at the 2′ position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6′ position and the A277S/T281V double mutant completely abolished the binding ability. A β3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.     

Probing functional interfaces of rod PDE γ-subunit using scanning fluorescent labeling

In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic α- and β-subunits (Pαβ) is inhibited by two γ-subunits (Pγ). On light stimulation of the photoreceptor cells, the GTP-bound α-subunit of visual G-protein transducin (G_taGTP) displaces the Pγ-subunits from their inhibitory sites on Pαβ, leading to the effector enzyme activation. We designed a number of Pγ mutants, each with a single cysteine residue evenly distributed at a different position along the Pγ polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of Pαβ and G_ta with the fluorescently labeled Pγ mutants suggests two distinct functional interfaces of Pγ. The Pαβ/Pγ interface is formed essentially by the C-terminus of Pγ and by the N-terminal portion of the Pγ polycationic region, Pγ-24-45, whereas the Pγ/G_ta interface includes the C-terminal portion of Pγ-24-45 and the region surrounding Pγ Cys68. Such functional organization of Pγ may represent an important element for the PDE activation mechanism during transduction of visual signals.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.