Abstract
Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis.
Highlights
It has been shown that the reduction in the intracellular [K þ ] and plasma membrane potential (PMP) depolarization are a late event since involve inhibition of Na þ /K þ pump by caspase-mediated degradation of its b-subunit.[2,14]
Apoptosis is clearly associated with PMP depolarization,[2,14,29] while inhibition of apoptosis by Bcl-2 and Mcl-1 is associated with PMP hyperpolarization.[3,4]
The role of K þ channels in apoptosis has been studied extensively because it is thought to be a pathway for K þ exit from the cytoplasm,[5,6,24,29,30] which is a necessary step for the activation of caspases.[5,13,18]
Summary
It has been shown that the reduction in the intracellular [K þ ] and PMP depolarization are a late event since involve inhibition of Na þ /K þ pump by caspase-mediated degradation of its b-subunit.[2,14]. The activation of caspases requires the reduction of the [K þ ]i regardless the inducer of apoptosis used or cell type studied.[13,15,16,17] High external [K þ ] inhibits both the extrinsic (Fas-mediated) and the intrinsic (etoposideinduced) cell death programs apparently by inhibiting the release of cytochrome c (cyt c) from mitochondria.[17] it has been shown that staurosporine (STS)-induced cyt c release in both HeLa and neuroblastoma cells (SK-N-BE(2)) is not inhibited by avoiding reduction of [K þ ]i.16 It appears that high intracellular K þ protects against apoptosis by inhibiting the apoptosome assembly.[13,16,18] Apparently, the procaspase-3 activity is inhibited by high [K þ ] because its activity decrease to B50% in [K þ ] above 25 mM K, in contrast mature caspase-3 activity is unaltered by reducing [K þ ].18. Ion channel inhibitors while blocking activation of caspase-3 do not block STS-induced reduction in [K þ ]i implying that STS-induced K þ loss is not occurring by these ion channels activated by STS
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