Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel

The ATP-sensitive potassium (K(ATP)) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 K(ATP) channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (tau(o)) and the short closed time (tauC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer tau(o), shorter tauC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the K(ATP) channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.     

Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea

Interleukin-12 receptor beta 1 ( IL12RB1), interleukin-12 receptor beta 2 ( IL12RB2), and interferon gamma receptor 1 ( IFNGR1) perform important roles in the host defense against intracellular pathogens such as Mycobacteria. Several mutations within their genes have been confirmed as associated with increased susceptibility to mycobacterial infection. However, the association between mutations of the IL12RB1, IL12RB2, and IFNGR1 encoding genes and lepromatous leprosy has not been studied. This study screened for polymorphisms within IL12RB1, IL12RB2, and IFNGR1 encoding genes in the Korean populations using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) DNA sequencing assay, and an association study was performed using the missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), and 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14M), and 1443 T/C (L467P) for the IFNGR1 encoding genes. There were no differences in the genotype and allele frequencies of IL12RB1 and IFNGR1 genes between 93 lepromatous leprosy patients and 94 control subjects. In conclusion, missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14 M), and 1443 T/C (L467P) of the IFNGR1 encoding genes have no association with the susceptibility to lepromatous leprosy in the Korean population.     

Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously.

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.     

Dinitropyrenes induce gene mutations in multiple organs of the lambda/lacZ transgenic mouse (Muta Mouse)

Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust. DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations. To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human. For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs. A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks. Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis. The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E. coli. In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E. coli. Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis. Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene. Three-four-fold increases were observed in stomach for both genes. A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene. The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice. Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups. The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%). The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule). The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo.     

Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop

Protease-activated receptor 1 (PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (G(q/11), G(i/o), and G(12/13)) and their linked signaling pathways to regulate a broad range of pathophysiological processes. However, the molecular mechanisms whereby PAR1 interacts with multiple G proteins are not well understood. Whether PAR1 interacts with various G proteins at the same, different, or overlapping binding sites is not known. Here we investigated the functional and specific binding interactions between PAR1 and representative members of the G(q/11), G(i/o), and G(12/13) subfamilies. We report that G(q/11) physically and functionally interacts with specific amino acids within the second intracellular (i2) loop of PAR1. We identified five amino acids within the PAR1 i2 loop that, when mutated individually, each markedly reduced PAR1 activation of linked inositol phosphate formation in transfected COS-7 cells (functional PAR1-null cells). Among these mutations, only R205A completely abolished direct G(q/11) binding to PAR1 and also PAR1-directed inositol phosphate and calcium mobilization in COS-7 cells and PAR1-/- primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either G(o) or G(12), or their functional coupling to linked pertussis toxin-sensitive ERK phosphorylation and C3 toxin-sensitive Rho activation, respectively. In astrocytes, our findings suggest that PAR1-directed calcium signaling involves a newly appreciated G(q/11)-PLCε pathway. In summary, we have identified key molecular determinants for PAR1 interactions with G(q/11), and our findings support a model where G(q/11), G(i/o) or G(12/13) each bind to distinct sites within the cytoplasmic regions of PAR1.     

Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within activin receptor 2 (ACVR2)

We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.     

Kinetic studies and structural models of the association of E. coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates

The kinetics of interaction of Esigma(70) RNA polymerase (R) with the lambdaP(R) promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42 degrees C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I(2), RP(o)) are single-exponential with first order rate constant beta(CR). Interpretation of the polymerase concentration dependence of beta(CR) in terms of the three step mechanism of open complex formation yields the equilibrium constant K(1) for formation of the first kinetically significant intermediate (I(1)) and the forward rate constant (k(2)) for the conformational change converting I(1) to the second kinetically significant intermediate I(2): R + P-->(K(1))<--I(1)(k(2))-->I(2). Use of rapid quench mixing allows K(1) and k(2) to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60 bp) interface formed in I(1), its relatively small binding constant K(1) at 37 degrees C at this [salt] (approximately 6 x 10(6) M(-1)) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k(a)[DeltaC(o,++)(a)= -1.5(+/-0.2)kcal K(-1)], now shown to originate directly from formation of I(1) [DeltaC(o)(1)= -1.4(+/-0.3)kcal K(-1)] rather than from the formation of I(2) as previously proposed. The isomerization I(1)-->I(2) exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy [E(act)(2)= 34(+/-2)kcal]. This kinetic signature suggests that formation of the transition state (I(1)-I(2)++ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I(1) involves coupled folding of unstructured regions of polymerase (beta, beta' and sigma(70)) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of sigma(70) and possibly domain 1 of beta induce a kink at the -11/-12 base pairs of the lambdaP(R) promoter which places the downstream DNA (-5 to +20) in the jaws of the beta and beta' subunits of polymerase in I(1). These early interactions of beta and beta' with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.     

Mg(2+) binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

BK channels are activated by intracellular Ca(2+) and Mg(2+) as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca(2+) sites, one low-affinity Mg(2+) site, and a voltage sensor. This study further investigates the mechanism of Mg(2+) activation by using single-channel recording to determine separately the action of Mg(2+) on the open and closed states of the channel. To limit Mg(2+) action to the Mg(2+) sites, the two high-affinity Ca(2+) sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg(2+) decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg(2+) can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg(2+) action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (P(o)) is dramatically increased by 10 mM Mg(2+) when voltage sensors are activated with either depolarization or the mutation R210C. The increased P(o) arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg(2+) has no detectable effects on P(o) or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg(2+) can bind to and alter the gating of both closed and open states to increase P(o), provided that one or more voltage sensors are activated.     

Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene

Alteration in hepatitis B virus (HBV) secretion efficiency may have pathological consequences. Naturally occurring mutations that regulate virion secretion have not been defined. We recently identified HBV genomes displaying high (4B), substantially reduced (3.4), or negative (4C) virion secretion. In the present study, the underlying mutations were mapped. A T552C point mutation in the 4B genome was responsible for its enhanced virion secretion, whereas a G510A mutation in 3.4 and G660C in 4C impaired virus secretion. The three point mutations generate M133T, G119E, and R169P substitutions in the S domains of viral envelope proteins, respectively, without modifying the coding capacity of the overlapping polymerase gene. The mutated residues are predicted to lie in the luminal side of the endoplasmic reticulum (ER) or to be embedded in the ER membrane and thus are not involved in contact with core particles during envelopment. Of the two mutations inhibitory of virion secretion, G510A greatly reduced small envelope protein (hepatitis B surface antigen [HBsAg]) levels both inside cells and in culture medium, whereas G660C specifically abolished HBsAg secretion. Surprisingly, a T484G mutation in the 4B genome, generating an I110M substitution in the S domain, could also reduce HBsAg secretion and block virion secretion. However, its inhibitory effect was suppressed in the 4B genome by the T552C mutation, the enhancer of virion secretion. T552C can also override the inhibitory G510A mutation, but not the G660C mutation. These findings suggest a hierarchy in the regulation of virion secretion and a close link between defective virion secretion and impaired HBsAg formation or secretion.     

ELF electromagnetic fields increase hydrogen peroxide (H2O2)-induced mutations in pTN89 plasmids

We have examined the mutational effects of hydrogen peroxide (H(2)O(2)) in the presence and absence of an extremely low-frequency magnetic field (ELFMF), using pTN89 plasmids. Mutations were detected in the supF gene carried by these plasmids in Escherichia coli. The plasmids were either treated with H(2)O(2) (1microM) alone at 37 degrees C for 4h, or were exposed to an ELFMF (60Hz, 5millitesla (mT)) simultaneously with H(2)O(2) treatment. The mutation frequency was 2.28 x 10(-4) for H(2)O(2) treatment alone, and 5.81 x 10(-4) for ELFMF exposure with H(2)O(2) treatment. We did not observe any mutations using treatment with ELFMF exposure alone. This indicates that the ELFMF may potentiate H(2)O(2)-induced mutation. Sequence analysis of the supF mutant plasmids revealed that base substitutions, G: C-->A :T transitions and G:C-->T:A transversions were dominant in both treatment groups, and there was no difference in the mutation spectrum or the hotspots between the groups. Therefore, ELFMFs may interact and potentiate the damage induced by H(2)O(2), resulting in an increase in the number of mutations.     

Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.     

Studies of type I collagen (COL1A1) alpha1 chain in patients with osteogenesis imperfecta

Nucleotide sequences of exon 51, adjacent intron areas, and regulatory region of the alpha1 chain of type I collagen (COL1A1) gene were analyzed in 41 patients with osteogenesis imperfecta (OI) from 33 families and their 68 relatives residing at Bashkortostan Republic (BR). Six mutations (four nonsense mutations c.967G > T (p.Gly323X), c.1081C > T (p.Arg361X), c.1243C > T (p.Arg415X), and c.2869C > T (p.Gln957X)) in patients of the Russian origin and two mutations with open reading frame shift c.579delT (p.Gly194ValfsX71), and c.2444delG (p.Gly815AlafsX293)) in patients with OI of Tatar ethnicity as well as 14 single nucleotide polymorphisms in the COL1A1 gene were revealed. Mutations c.967G > T (p.Gly323X) and three alterations in the nucleotide sequence c.544-24C > T, c.643-36delT, and c.957 + 10insA were described for the first time.     

Site-directed mutations in the third intracellular loop of the serotonin 5-HT(1A) receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.     

Four New Mutations and Two Polymorphic Variants of the Low-Density Lipoprotein Receptor Gene in Familial Hypercholesterolemia Patients from St. Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas. To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively. These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Four new mutations and polymorphic variants of the low density lipoprotein receptor in patients with familial hypercholesterolemia in Saint Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.     

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.