By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Immunofluorescent staining of mAno1 containing tandem hemaglutinin (HA) epitopes inserted at various locations. A, HA tags were inserted into mAno1-EG...Show More
Immunofluorescent staining of mAno1 containing tandem hemaglutinin (HA) epitopes inserted at various locations. A, HA tags were inserted into mAno1-EGFP at amino acids 570, 614, 672, 700, and 824. After transient expression, nonpermeabilzed intact cells were stained with antibody for HA epitope. Green: Ano1-GFP; red: anti-HA and merged image. Duplicate cover slips were permeabilized before incubation with HA antibody (red: permeabilized, anti-HA). For each construct, images were acquired at the same gain and settings, but settings may differ between constructs that were imaged on different days. Raw images from the Zeiss Zen acquisition software from permeabilized and nonpermeabilized cells were assembled in Adobe Photoshop CS5 and brightness-adjusted and contrast-adjusted for all four panels equally. Anoctamin-1 (Ano1) currents for each construct were recorded with 20 μmol/L Ca. Average peak amplitude at +100 mV and the number of recorded cells are listed. B, Topological models of mAno1. The locations of HA tags are indicated with red numbers. Left, Re-entrant loop model. Right, revised model. The topology of the sequence depicted in gray remains in question.Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Mutation of two critical amino acids, E702 and E705, dramatically affects Ca2+-gated but not voltage-gated current of mAno1. Representative whole-cell...Show More
Mutation of two critical amino acids, E702 and E705, dramatically affects Ca2+-gated but not voltage-gated current of mAno1. Representative whole-cell recordings of anoctamin-1 (Ano1) current in transfected HEK293 cells at the indicated free [Ca2+]. Voltage protocol is shown above (A). A, WT-mAno1. B, E702Q/E705Q mutant mANO1. Steady-state current–voltage (I–V) relationships for (C) WT-mAno1 and (D) E702Q/E705Q mANO1with different [Ca]i (N=5–9). E, I–V relationships for E702D, E702K, E705D, and E705K mutants with 20 μmol/L Ca2+ (N=5–9).Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Activation and deactivation kinetics of anoctamin-1 (Ano1) with rapid Ca 2+ perfusion in inside-out excised patches. Representative traces of Ano1 cur...Show More
Activation and deactivation kinetics of anoctamin-1 (Ano1) with rapid Ca 2+ perfusion in inside-out excised patches. Representative traces of Ano1 current in response to application (left) and washout (right) of Ca2+ at the indicated holding potentials. A, WT-mAno1. B, E702Q mANO1. C, E705Q mANO1. D–F, Vm dependence of τon, τoff, and EC50 for WT Ano1 (open circles), E702Q (filled circles), and E705Q (filled triangles).Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Effects of sulfhydryl modification on activation and deactivation kinetics of anoctamin-1 (Ano1) in excised inside-out patches with rapid Ca2+ perfusi...Show More
Effects of sulfhydryl modification on activation and deactivation kinetics of anoctamin-1 (Ano1) in excised inside-out patches with rapid Ca2+ perfusion. Excised patches were switched between zero and high Ca2+. They were then exposed to MTS reagent in the presence of zero Ca2+ for 10 seconds, MTS reagent was washed away, and the patch was switched from zero Ca2+ to high Ca2+ again. Normalized current traces of E702C mAno1 showing examples of changes in τon and τoff caused by MTSET+ (A) and MTSES− (B). Traces were normalized to the same maximal amplitude. As shown in Online Figure III, MTSET+ causes a decrease in current and MTSES− increases the current. The magnitude of the effect of MTS reagent on current amplitude depends on where on the Ca2+dose–response curve the experiment is performed. C–E, Lack of effect of MTS reagents on τon, τoff, and EC50 for WT Ano1. F–H, Effects of MTS reagents on τon, τoff, and EC50 for E702C mAno1. Open circle: before application of MTS reagent. Filled circle: after MTSET+. Triangle: after MTSES−. N=3–6.Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Effect of R621E mutation on anion:cation permeability of mAno1. Whole-cell recordings of (A) WT mAno1 in symmetrical 150 mmol/L NaCl with 180 nmol/L C...Show More
Effect of R621E mutation on anion:cation permeability of mAno1. Whole-cell recordings of (A) WT mAno1 in symmetrical 150 mmol/L NaCl with 180 nmol/L Cai and (B) R621E mAno1 in symmetrical 150 mmol/L NaCl with 1.1 μmol/L Cai. The R621E mutation decreased the Ca2+ sensitivity of the channel, requiring a larger Ca2+ concentration to generate a measurable current. Current–voltage relationships of (C) WT mAno1 and (D) R621E mAno1 with different extracellular [NaCl]. E, Change in reversal potential (ΔErev) for different extracellular [NaCl] or [CsCl] determined from experiments like those in (C) and (D). Lines are best fits to the Goldman-Hodgkin-Katz equation.Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Essential cysteines in mAno1 and scanning cysteine accessibility of the proposed new transmembrane domain 6 (amino acids 620–646). A, Six extracellula...Show More
Essential cysteines in mAno1 and scanning cysteine accessibility of the proposed new transmembrane domain 6 (amino acids 620–646). A, Six extracellular cysteines are required for mAno1 function. Amplitudes of whole-cell currents activated by 20 μmol/L [Ca2+]i at +100 mV from WT and cysteine-substituted mutants of mAno1 expressed in HEK293 cells. Inset shows location of cysteines (solid red circles: essential; open red circles: not essential for channel function) in the old model of mAno1. mAno1 with all cysteines (C) except C370, C379, C383, C386, C395, and C836 replaced with serines. C836S is wild-type (WT) mAno1, with C836 only replaced with serine. The other constructs have each of the essential cysteines in mAno16C as indicated replaced with serine. B, Example of change in current amplitude of G629C mAno16C. MTSET-induced increase in current is not reversed by washout of MTSET but is reversed by 5 mmol/L DTT. C, Average anoctamin-1 (Ano1) currents recorded from WT mAno1, mAno16C, and cysteine-substituted mAno16C. D, Effects of MTSET+ (left), MTSES− (middle), and MTSEA+ (right) on cysteine-substituted mAno16C. The percent change of current at +100 mV within 2 minutes of exposure to MTS reagent was calculated by [100 (IafterMTS−IbeforeMTS)/IbeforeMTS]. Cysteine-substituted amino acids labeled with an asterisk (*) were significantly (P<0.05) affected by MTS reagents (N=3–6).Show Less
By KuaiYu, CharityDuran, ZhiqiangQu, Yuan-YuanCui and H. CrissHartzell
Effects of replacement of amino acids in mAno16C with cysteine on I− permeability and conductance relative to Cl−. Relative I− permeability was calcul...Show More
Effects of replacement of amino acids in mAno16C with cysteine on I− permeability and conductance relative to Cl−. Relative I− permeability was calculated from the shift in reversal potential after replacing extracellular Cl− with I−. Relative I− conductance was measured as the ratio of slopes of the current–voltage relationship at the reversal potential after and before extracellular anion change (N=3–10).Show Less
Robert Rovetti, Xiaohua Cui, Alan Garfinkel, James N. Weiss, Zhilin Qu
Circulation Research May 2010, 106 (10) 1582-1591; DOI: https://doi.org/10.1161/CIRCRESAHA.109.213975
By RobertRovetti, XiaohuaCui, AlanGarfinkel, James N.Weiss and ZhilinQu
Figure 1. The spatially distributed Ca cycling model. A, Schematic plots (side view and top view) of a coupled CRU network. B, Detailed illustration o...Show More
Figure 1. The spatially distributed Ca cycling model. A, Schematic plots (side view and top view) of a coupled CRU network. B, Detailed illustration of a CRU. C, Simplified Markov model of the LCC. “O” is the open state, and “I” and “C” are the closed states. The LCC is activated by voltage and inactivated by voltage and Ca (as indicated by “Ca” in the graph) in the dyadic space. D, The Markov model of the RyR from Stern et al.27 “O” is the open state; “I”, “R”, and “C” are the closed states. RyR is activated and inactivated by Ca (as indicated by “Ca” in the graph) in the dyadic space.Show Less
Figure 1. The spatially distributed Ca cycling model. A, Schematic plots (side view and top view) of a coupled CRU network. B, Detailed illustration o...Show More
