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• Articles by A.B. Harkins
• Articles by S.M. Baylor

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• AM-loading of fluorescent Ca2+ indicators into intact single...

  AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle Biophysical Journal, Volume 72, Issue 6, 1 June 1997, Pages 2736-2747 M. Zhao, S. Hollingworth and S.M. Baylor Abstract The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i. Abstract | PDF (1187 kb)

• Properties of tri- and tetracarboxylate Ca2+ indicators in f...

  Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers Biophysical Journal, Volume 70, Issue 2, 1 February 1996, Pages 896-916 M. Zhao, S. Hollingworth and S.M. Baylor Abstract Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed. Abstract | PDF (2285 kb)

• Conductance and permeation of monovalent cations through dep...

  Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells Biophysical Journal, Volume 71, Issue 2, 1 August 1996, Pages 787-794 A. Lepple-Wienhues and M.D. Cahalan Abstract We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model. Abstract | PDF (712 kb)

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Abstract

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36–40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02–0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2–0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.