Intracellular K+ concentration decrease is not obligatory for apoptosis

Abstract

K(+) efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K(+) and subsequent reduction in ionic strength are suggested to release the inhibition of proapoptotic caspases. In this work, a new K(+)-specific microelectrode was used to study possible alterations in intracellular K(+) in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K(+) was verified with parallel electrophysiological measurements. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K(+) concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K(+) was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel-expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K(+).

FIGURE 1.

Representative K⁺ current recordings (upper) and corresponding I(V) curves (lower) measured electrophysiologically in Kv channel-expressing Xenopus oocytes. A, data for control oocytes. B–D, data for oocytes injected with the indicated test solutions. The holding potential was set to −80 mV, and test pulses ranged from −80 to +50 mV. The current generated by stepping to 0 mV is marked in red in each recording.

FIGURE 2.

Electrophysiological recordings and the K⁺-selective microelectrodes report similar intracellular K⁺ concentrations. A, scanning electrode image of the K⁺-selective microelectrode before intracellular measurements. B, intracellular K⁺ concentrations in Kv channel-expressing Xenopus oocytes measured with two-electrode voltage-clamp (TEVC) and K⁺-selective microelectrode techniques. Data are expressed as mean values for control oocytes and oocytes injected with 50 nl of indicated test solutions. Error bars show S.E. n = 3–5. C, scanning electron microscope image of the K⁺-selective microelectrode after intracellular measurements.

FIGURE 3.

Caspase-3 activity in oocytes not expressing Kv channels. Measurements were done in control solution (n = 4) and after 3-h (n = 4) and 6-h (n = 3) exposure to 1 μm STS. The fluorescence after caspase-3 cleavage of Ac-DEVD-AMC was measured with photospectrometry and corrected with total protein level. Data show mean values ± S.E. (error bars).

FIGURE 4.

Intracellular K⁺ concentrations in apoptotic Xenopus oocytes with and without dense expression of Kv channels. A, intracellular concentration of K⁺ after 6-h incubation in STS. Mean values for oocytes densely expressing Kv channels (RNA injected) were measured with the two-electrode voltage-clamp method (TEVC; n = 5) or K⁺-selective microelectrode method (n = 5). Intracellular K⁺ concentration in oocytes not expressing Kv channels (Not RNA injected) was measured with the K⁺-selective microelectrode (n = 5). Data are expressed as mean values ± S.E. (error bars). B, intracellular K⁺ concentrations measured with electrophysiological method. Measurements were made in control solution (n = 5) and after 1 (n = 3), 2 (n = 3), 3 (n = 3), and 6 h (n = 5) of 1 μm STS exposure.

FIGURE 5.

Caspase-3 activity in apoptotic oocytes expressing (RNA injected) or not expressing (Not RNA injected) Kv channels. The fluorescence after caspase-3 cleavage of Ac-DEVD-AMC was measured with photospectrometry and corrected with total protein level. Data show mean values ± S.E. (error bars).

FIGURE 6.

Intracellular K⁺ concentrations in control and apoptotic Xenopus oocytes with dense expression of mutant Shaker Kv channels. R365C was measured with the two-electrode voltage-clamp method (TEVC) and the nonconducting W434F mutant with the K⁺-selective microelectrode method. STS-treated cells were incubated for 6 h in 1 μm STS. Data are expressed as mean values ± S.E. (error bars).