A new generation of Ca2+ indicators with greatly improved fluorescence properties.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.

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Cytoplasmic pH and free Mg2+ in lymphocytes.

Measurement of Ca2+ concentrations in living cells

1. The Na-Ca exchange current was investigated in single ventricular cells from guinea-pig hearts by combining the techniques of whole-cell voltage clamp and intracellular perfusion. 2. The membrane conductance was minimized by blocking Ca and K channels as well as the Na-K pump. Under these conditions, when Ca2+ was loaded internally by a pipette solution containing 430 nM-Ca2+, changing the Li+-rich external solution to a Na+-rich one induced a significant inward current. Applying external Na+ in the absence of internal Ca2+ did not appreciably change the current. 3. In contrast, perfusing 1 mM-external Ca2+ in the presence of internal Na+ which was loaded by a 20 mM-Na+ pipette solution, induced a marked outward current. Ca2+ superfusion in the absence of internal Na+ caused only a small current change. 4. The current-voltage relation of external-Ca2+- and external-Na+-induced current showed almost exponential voltage dependence as given by the equation i = a exp (rEF/RT), where a is a scaling factor that determines the magnitude of the current and r is a partition parameter used in the rate theory and represents the position of the energy barrier in the electrical field, which indicates the steepness of the voltage dependence of the current. E, F, R and T have their usual meanings. The value of a was 1-2 microA/microF and r about 0.35 for the Ca2+-induced outward current. At very positive or negative potentials, the current magnitude became smaller than expected from an exponential relation. 5. The current was blocked by heavy metal cations, such as La3+, Cd2+, Mn2+ and Ni2+ and partially blocked by amiloride and D600. 6. The temperature coefficient (Q10) value of the Ca2+-induced outward current was 3.6 +/- 0.4 (n = 4) at 0 mV and 4.0 +/- 0.9 at 50 mV in the range between 21 and 36 degrees C. 7. The outward current magnitude showed a sigmoidal dependence upon the external Ca2+ concentration with a half-maximum concentration, K1/2 of 1.38 mM and a Hill coefficient of 0.9 +/- 0.2 (n = 5). 8. Sr2+ could replace Ca2+ with K1/2 of 7 mM. Mg2+ and Ba2+, however, did not replace Ca2+. 9. The inward current component also showed a sigmoidal external Na+ dependence with K1/2 of 87.5 +/- 10.7 mM and a Hill coefficient of 2.9 +/- 0.4 (n = 6). 10. The reversal potential of the current was obtained near the values expected for 3 Na+:1 Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig.

The inwardly rectifying K channel provides the resting K conductance in a variety of cells1–4. This channel acts as a valve or diode, permitting entry of K+ under hyperpolarization, but not its exit under depolarization. This behaviour, termed inward rectification, permits long depolarizing responses which are of physiological significance for the pumping function of the heart and for fertilization of egg cells5. Little is known about the outward currents through the inwardly rectifying K channel, despite their great physiological importance, and the mechanism of inward rectification itself is unknown. We have used improved patch clamp techniques to control the intracellular media, and have recorded the outward whole-cell and single-channel currents. We report here that the channel conductance is ohmic and that the well-known inward rectification of the resting K conductance is caused by rapid closure of the channel accompanied by a voltage-dependent block by intracellular Mg2+ ions at physiological concentrations.

R.Y. Tsien

Papers

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium