Roles of L-type Ca^(2+) Channel Subunits of Skeletal Muscle in Dihydropyridine Binding : Evidence for Functional Interaction among α₁, β, γ and α₂δ Subunits

Suh, Haeyoung,birnbaumer, Lutz 아주대학교 의과학연구소 1996 아주의학 Vol.1 No.1

Purified skeletal muscle 1,4-dihydropyridine (DHP) receptois are composed of five polypeptides termed α₁, β, γ, α₂, and 5. Among these, the α₁ subunit is known to be sufficient to function as a voltage-dependent Ca^(2+) channel and a DHP receptor. The α₁ alone exhibits very similar allosteric regulation of DHP binding to that found in skeletal muscle T-tubules. However, we previously showed that in the absence of Ca^(2+), DHP binding to α₁ alone at subsaturating concentrations was reduced, which could be restored by the (-) stereoisomer of a phenylalkylamine, D600. We hypothesized that this difference is due to lack of other regulatory subunits, specifically the β, γ and α₂δ components that copurify with α₁. In order to test our hypothesis, we coexpressed the α₁ subunit with non-α₁ components in mouse fiboblasts, L cells, and monkey kidney cells, COS.M6 in various combinations-and compared their DHP binding activity for the effect of (-)D600 in the absence of Ca^(2+). Coexpression of 13 with α₁ did not normalize the abnormal enhancing effect of (-)D600 on DHP binding. Coexpression of either γ or α₂δ, partially reduced the enhancing effect of (-)D600. Importantly, coexpression of all the components, that is, when the receptor was composed of α₁β γα₂δ, completely abrogated the abnormal effect of (-)D600. Thus, our data clearly demonstrate that all the component copurifying with α₁ are essential to constitute the functional DHP receptor.

Simultaneous deletion of floxed genes mediated by CaMKIIa-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goa

최찬일,윤상필,최정미,김성수,이영돈,Lutz Birnbaumer,서해영 생화학분자생물학회 2014 Experimental and molecular medicine Vol.46 No.-

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIa) promoter limits expression to specific regions of the forebrainand thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIa-Cre can be utilized forsimultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26þ/stop-lacZ::CaMKIIa-Creþ/Cre mice generated by crossing CaMKIIa-Creþ/Cre mice with floxed ROSA26 lacZ reporter (Rosa26þ/stop-lacZ) miceexhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% ofthe offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate thatrecombination can occur in the germ cells of male Rosa26þ/stop-lacZ::CaMKIIa-Creþ/Cre mice. Similarly, when double transgenicGnaoþ/f::CaMKIIa-Creþ/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately22% of the offspring carried the disrupted allele (GnaoD) without inheriting the Cre transgene. The GnaoD/D mice closelyresembled conventional Go-alpha knockout mice (Gnao/) with respect to impairment of their behavior. Thus, we concludethat CaMKIIa-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brainand for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIa-Cremice as breeding pairs.

Resistance to pathologic cardiac hypertrophy and reduced expression of CaV1.2 in Trpc3-depleted mice

Han, Jung Woo,Lee, Young Ho,Yoen, Su-In,Abramowitz, Joel,Birnbaumer, Lutz,Lee, Min Goo,Kim, Joo Young Springer-Verlag 2016 MOLECULAR AND CELLULAR BIOCHEMISTRY - Vol.421 No.1

<P>Sustained elevation of intracellular Ca2+ concentration ([Ca2+](i)) reprograms cardiovascular cell fate, leading to cellular hypertrophy via Ca2+-calmodulin/calcineurin (Cn)/NFAT activation. Accumulating evidence suggests that transient receptor potential canonical (Trpc) channels play important roles in the development of pathologic cardiac hypertrophy. Here, we demonstrated that Trpc3 mediates pathologic cardiac hypertrophy in neurohumoral elevation via direct regulation of Ca(V)1.2 expressions. Elevated PE (phenylephrine) was maintained in mice by continuous infusion using an osmotic pump. Wild-type (WT) mice, but not Trpc3 (-/-) showed a sudden decrease in blood pressure (BP) or death following elevation of BP under conditions of elevated PE. Trpc3 (-/-) mesenteric artery showed decreased PE-stimulated vasoconstriction. Analysis of morphology, function, and pathologic marker expression revealed that PE elevation caused pathologic cardiac hypertrophy in WT mice, which was prevented by deletion of Trpc3. Interestingly, protection by Trpc3 deletion seemed to be a result of reduced cardiac Ca(V)1.2 expressions. Basal and PE induced increased expression of protein and mRNA of Ca(V)1.2 was decreased in Trpc3 (-/-) heart. Accordingly, altered expression of Ca(V)1.2 was observed by knockdown or stimulation of Trpc3 in cardiomyocytes. These findings suggest that Trpc3 is a mediator of pathologic cardiac hypertrophy not only through mediating part of the Ca2+ influx, but also through control of Ca(V)1.2 expressions.</P>

Simultaneous deletion of floxed genes mediated by CaMKIIα-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goα

Choi, Chan-Il,Yoon, Sang-Phil,Choi, Jung-Mi,Kim, Sung-Soo,Lee, Young-Don,Birnbaumer, Lutz,Suh-Kim, Haeyoung Nature Publishing Group 2014 Experimental and molecular medicine Vol.46 No.5

<P>The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26<SUP>+/stop-lacZ</SUP>::CaMKIIα-Cre<SUP>+/Cre</SUP> mice generated by crossing CaMKIIα-Cre<SUP>+/Cre</SUP> mice with floxed ROSA26 lacZ reporter (Rosa26<SUP>+/stop-lacZ</SUP>) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26<SUP>+/stop-lacZ</SUP>::CaMKIIα-Cre<SUP>+/Cre</SUP> mice. Similarly, when double transgenic Gnao<SUP>+/f</SUP>::CaMKIIα-Cre<SUP>+/Cre</SUP> mice carrying a floxed <I>Go-alpha</I> gene (Gnao<SUP>f/f</SUP>) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao<SUP>Δ</SUP>) without inheriting the Cre transgene. The Gnao<SUP>Δ/Δ</SUP> mice closely resembled conventional Go-alpha knockout mice (Gnao<SUP>−/−</SUP>) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.</P>

Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

Seo, Kinya,Rainer, Peter P.,Shalkey Hahn, Virginia,Lee, Dong-ik,Jo, Su-Hyun,Andersen, Asger,Liu, Ting,Xu, Xiaoping,Willette, Robert N.,Lepore, John J.,Marino Jr., Joseph P.,Birnbaumer, Lutz,Schnackenb National Academy of Sciences 2014 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.111 No.4

<P>Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC<SUB>50</SUB>, 3–21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.</P>

Development of the main olfactory system and main olfactory epithelium-dependent male mating behavior are altered in G_o-deficient mice

Choi, Jung-Mi,Kim, Sung-Soo,Choi, Chan-Il,Cha, Hye Lim,Oh, Huy-Hyen,Ghil, Sungho,Lee, Young-Don,Birnbaumer, Lutz,Suh-Kim, Haeyoung National Academy of Sciences 2016 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.113 No.39

<P>In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein G(o) is widely expressed in the MOE and VNO of mice. Early studies indicated that G(o) expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of G(o) in the MOE have remained poorly defined. Here, we examined the role of G(o) in the MOE using mice lacking the a subunit of G(o). Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that G(o) signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.</P>