Characterizing the Role of Thr352 in the Inhibition of the Large Conductance Ca 2ϩ -Activated K ϩ Channels by 1-[1-Hexyl-6- (methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone

ABSTRACT Large conductance Ca 2ϩ -activated K ϩ (BK) channels are known to be regulated by both intracellular Ca 2ϩ and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and activators. Using both whole-cell and single-channel electrophysiological studies we have characterized the possible role that a threonine residue in the pore region of the channel has on function and interaction with BK channel modulators. A threonine-to-serine substitution at position 352 (T352S) resulted in a 59-mV leftward shift in the voltage-dependent activation curve. Single-channel conductance was 236 pS for the wild-type channel and 100 pS for the T352S mutant, measured over the range Ϫ80 mV to ϩ80 mV. In addition, there was an almost 10-fold reduction in the potency of the BK channel inhibitor 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP), the IC 50 values being 4.3 Ϯ 0.3 and 38.2 Ϯ 3.3 nM for wild-type and mutant channel, respectively. There was no significant difference between wild type and the mutant channel in response to inhibition by iberiotoxin. The IC 50 was 8.1 Ϯ 0.3 nM for the wild type and 7.7 Ϯ 0.3 nM for the mutant channel. Here, we have identified a residue in the pore region of the BK channel that alters voltage sensitivity and reduces the potency of the blocker HMIMP

The Calcium Channel Gamma6 Subunit Analysis of Function and Determination of a Sequence Motif Critical for Its Effect

106 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The auxiliary gamma6 subunit of voltage dependent calcium channels is known to decrease calcium current density when co-expressed with the pore forming Cav3.1 subunit. In this study, I showed that Cav3.1 calcium channels are a major pathway for calcium influx at resting membrane potential, and that the gamma6 subunit is a modulator of this LVA window current in HEK-Cav3.1 cells by employing a calcium imaging technique. Further, I demonstrated that co-expression of gamma6 subunit decreases Cav2.3 dependent calcium current density. Previous studies using chimeric gamma subunits indicate that the N-terminal region, including the first transmembrane domain (TM1), is critical for the inhibitory function of the gamma6 subunit. In this study, I have investigated the functional properties of the gamma6 TM1 and identified critical motifs and residues in this region. Calcium current density in HEK-Cav3.1 cells was monitored following transfection with plasmids containing either gamma 6 TM1 or various mutants of this peptide. The gamma6 TM1 significantly inhibits the expression of Cav3.1 current suggesting that gamma 6 TM1 is both necessary and sufficient to inhibit Cav3.1 calcium current. Co-immunoprecipitation experiments indicate the presence of a gamma 6/alpha1 subunit complex when the two proteins are co-expressed. We identified two adjacent GxxxA motifs (G42xxxA46xxG 49xxxA53) in the gamma6 TM1 which are predicted to produce a long groove on one face of the helix due to the short side chains of the glycine and alanine residues. GxxxA and related motifs are thought to be important for promoting and stabilizing helix-helix interactions. Therefore we performed site directed mutagenesis of these specific residues in TM1 replacing the G and A residues with amino acids containing large side chains. The G42L and A46I mutants are no longer inhibitory while the G49L mutant retained the inhibitory function of the wild type. Our results suggest that the first GxxxA motif within TM1 of gamma6 is critical for its ability to inhibit Cav3.1 calcium current. Overall, my study enhances our understanding of the function and structure of the gamma6 subunit, and provides a basic approach to design novel therapeutic agents to target calcium channels.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

The Calcium Channel Gamma6 Subunit Analysis of Function and Determination of a Sequence Motif Critical for Its Effect

106 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The auxiliary gamma6 subunit of voltage dependent calcium channels is known to decrease calcium current density when co-expressed with the pore forming Cav3.1 subunit. In this study, I showed that Cav3.1 calcium channels are a major pathway for calcium influx at resting membrane potential, and that the gamma6 subunit is a modulator of this LVA window current in HEK-Cav3.1 cells by employing a calcium imaging technique. Further, I demonstrated that co-expression of gamma6 subunit decreases Cav2.3 dependent calcium current density. Previous studies using chimeric gamma subunits indicate that the N-terminal region, including the first transmembrane domain (TM1), is critical for the inhibitory function of the gamma6 subunit. In this study, I have investigated the functional properties of the gamma6 TM1 and identified critical motifs and residues in this region. Calcium current density in HEK-Cav3.1 cells was monitored following transfection with plasmids containing either gamma 6 TM1 or various mutants of this peptide. The gamma6 TM1 significantly inhibits the expression of Cav3.1 current suggesting that gamma 6 TM1 is both necessary and sufficient to inhibit Cav3.1 calcium current. Co-immunoprecipitation experiments indicate the presence of a gamma 6/alpha1 subunit complex when the two proteins are co-expressed. We identified two adjacent GxxxA motifs (G42xxxA46xxG 49xxxA53) in the gamma6 TM1 which are predicted to produce a long groove on one face of the helix due to the short side chains of the glycine and alanine residues. GxxxA and related motifs are thought to be important for promoting and stabilizing helix-helix interactions. Therefore we performed site directed mutagenesis of these specific residues in TM1 replacing the G and A residues with amino acids containing large side chains. The G42L and A46I mutants are no longer inhibitory while the G49L mutant retained the inhibitory function of the wild type. Our results suggest that the first GxxxA motif within TM1 of gamma6 is critical for its ability to inhibit Cav3.1 calcium current. Overall, my study enhances our understanding of the function and structure of the gamma6 subunit, and provides a basic approach to design novel therapeutic agents to target calcium channels.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

A novel in situ hydrothermal preparation route for Sb2S3 and its solar cell application

In this study, we reported a novel in situ hydrothermal approach for fabricating Sb2S3 films. Metal Sb films were first deposited on substrates using room-temperature radio frequency magnetron sputtering and then undergo an in situ transformation to Sb2S3 films through a novel low-temperature hydrothermal reaction. Characteristics of the obtained Sb2S3 films prepared by our method were investigated. Planar Sb2S3 solar cells utilizing in situ fabricated Sb2S3 film as absorber has also been fabricated and yielded a power conversion efficiency of 1.32%. (C) 2018 Elsevier B.V. All rights reserved

Effect of iron deficiency on c-kit⁺ cardiac stem cells in vitro.

AimIron deficiency is a common comorbidity in chronic heart failure (CHF) which may exacerbate CHF. The c-kit⁺ cardiac stem cells (CSCs) play a vital role in cardiac function repair. However, much is unknown regarding the role of iron deficiency in regulating c-kit⁺ CSCs function. In this study, we investigated whether iron deficiency regulates c-kit⁺ CSCs proliferation, migration, apoptosis, and differentiation in vitro.MethodAll c-kit⁺ CSCs were isolated from adult C57BL/6 mice. The c-kit⁺ CSCs were cultured with deferoxamine (DFO, an iron chelator), mimosine (MIM, another iron chelator), or a complex of DFO and iron (Fe(III)), respectively. Cell migration was assayed using a 48-well chamber system. Proliferation, cell cycle, and apoptosis of c-kit⁺ CSCs were analyzed with BrdU labeling, population doubling time assay, CCK-8 assay, and flow cytometry. Caspase-3 protein level and activity were examined with Western blotting and spectrophotometric detection. The changes in the expression of cardiac-specific proteins (GATA-4,TNI, and β-MHC) and cell cycle-related proteins (cyclin D1, RB, and pRB) were detected with Western blotting.ResultDFO and MIM suppressed c-kit⁺ CSCs proliferation and differentiation. They also modulated cell cycle and cardiac-specific protein expression. Iron chelators down-regulated the expression and phosphorylation of cell cycle-related proteins. Iron reversed those suppressive effects of DFO. DFO and MIM didn't affect c-kit⁺ CSCs migration and apoptosis.ConclusionIron deficiency suppressed proliferation and differentiation of c-kit⁺ CSCs. This may partly explain how iron deficiency affects CHF prognosis

A critical GxxxA motif in the γ6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current

The eight members of the calcium channel γ subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ2, γ3, γ4 and γ8 (the TARPs) modulate AMPA receptor localization and function, the γ1 and γ6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the γ6 subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of γ6 subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of γ6 is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix–helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of γ6 with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of γ6 reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the γ6 calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density

Isolation and identification of c-kit⁺ CSCs.

<p>(A) The c-kit⁺ CSCs isolated by immunomagnetic microbeads were almost small, round, phase-bright on the first day. (B) Three days later, c-kit⁺ CSCs gradually attached to the plate, proliferated, and clustered. (C) Quantitative analysis of surface markers of cells expanded in culture by FACS. (D) The expression of c-kit was examined with western blotting in the isolated cells (CSC) and cardiac myocytes (CM). (E) Immunostaining of isolated cells. All experiments were repeated 5 times.</p

Effect of iron deficiency on c-kit⁺ CSCs apoptosis.

<p>(A, B, C) Cellular apoptosis was analyzed. Cells were treated with DFO, MIM, H₂O₂, or the complex of DFO and Fe(III) for 24 h or 48 h. H₂O₂ was used as positive control. DFO, MIM, and the complex of DFO and Fe(III) didn’t induce cellular apoptosis. Then, caspase-3 expression (D, E, F, G) and activity (H, I) were explored. DFO, MIM, and the complex of DFO and Fe(III) didn’t regulate caspase-3 expression and activity. * p<0.05, ** p<0.01 (compared with control group). All experiments were repeated 5 times.</p