4-aminopyridine, a Kv channel antagonist, prevents apoptosis of rat cerebellar granule neurons.
Compelling evidence indicates that excessive potassium (K+) efflux and intracellular K+ depletion are the key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granule neurons induced by incubation in low-K+ (5 mM) and serum-free medium was associated with an increase in A-type transient inactivation of K+ channel current (IA) amplitude and modulation of channels' gating properties. Here, we showed that a classic K+ channel blocker, 4-aminopyradine (4-AP), significantly inhibited IA amplitude in a concentration-dependent manner (reduction of current by 10 microM and 10 mM 4-AP was 11.4+/-1.3% and 72.2+/-3.3%, respectively). Moreover, 4-AP modified the steady-state activation and inactivation kinetics of IA channels, such that the activation and inactivation curves were shifted to the right about 20 mV and 17 mV, respectively. Fluorescence staining showed that 4-AP dramatically increased the viability of cells undergoing apoptosis in a dose-dependent manner. That is, while 5 mM 4-AP was present, cell viability was 84.9+/-5.2%. Consistent with the cell viability analysis, internucleosomal DNA fragmentation by gel electrophoresis analysis showed that 5 mM 4-AP also protected against neuronal apoptosis. Furthermore, 4-AP significantly inhibited cytochrome c release and caspase-3 activity induced by low-K+/serum-free incubation. Finally, current-clamp analysis indicated that 5 mM 4-AP did not significantly depolarize the membrane potential. These results suggest that 4-AP has robust neuroprotective effects on apoptotic granule cells. The neuroprotective effect of 4-AP is likely not due to membrane depolarization, but rather that 4-AP may modulate the gating properties of IA channels in an anti-apoptotic manner.
Hu
Chang-Long
CL
Center for Brain Science Research, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 220 HanDan Road, Shanghai 200433, China.
Liu
Zheng
Z
Zeng
Xi-Min
XM
Liu
Zi-Qiang
ZQ
Chen
Xian-Hua
XH
Zhang
Zhi-Hong
ZH
Mei
Yan-Ai
YA
D015761:4-Aminopyridine / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000528:radiation effects; D009474:Neurons / Q000187:drug effects*; D018408:Patch-Clamp Techniques / Q000379:methods; D011188:Potassium / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D026902:Potassium Channel Blockers; D045304:Cytochromes c; D015761:4-Aminopyridine; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D011188:Potassium