Investigation On Neurotoxicity Of Amyloid β-protein: Effects Of AβP31-35 And AβP25-35 On K～+ Currents And [Ca～(2+)]_i In Rat Cerebral Cortical Neurons And Hippocampal Neurons

Excessive extracellular deposition of amyloid (J-protein (AJ3P) in brain areas that forms specific senile plaques and the degeneration of cortical and hippocampal neurons are the invariant characteristic features of Alzheimer's disease (AD). A0P is a polypeptide of 39-43 amino acids, and is generated by enzymatic cleavages from amyloid precursor protein. Up to now, it is generally accepted that the shorter fragment of AβP, AβP25-35, possesses all the biological activities of full-length AβP molecule, and recently, our lab and others have showed that AβP31-35, an up-date known shortest fragment of AβP, also possesses similar neurotoxicity and thus this peptide has been used as a tool in analysis the effects of AβP on hippocampal and cortical neurons.At the same time, the mechanisms underlying the AβP-mediated neurodegeneration are not well understood, though several hypotheses have been proposed, such as direct neurotoxicity, free radical production, [Ca2+]j overloading, formation of new ion channels and so on. In all these hypotheses, the role of K+ channels and its relationship to [Ca2+]j overloading have been recently emphasized.In present experiments, we observed the effects of AβP31-35 on the BKcurrents in rat cerebral cortical neurons by using inside-out patch clamp technique, and also the U and IK currents in rat hippocampal CA1 neurons by using whole-cell patch clamp technique; the effects of AβP31-35 on the intracellular Ca2+ concentration of hippocampal neurons were detected by fluorescence imaging method with neurons being incubated with Ca2+-sensitive dye Fura-2/AM. The effects of AβP25-35 were also observed with an attempt to prove whether AβP31-35 being equivalent to AβP25-35 in these kinds of experiments, and thus it could be used in analyzing the effects of the full-length molecules oPart I AβP31-35 and AβP25-35 suppress BK channels in rat cerebral cortical neuronsIn this study, we investigated the effects of AβP31-35 and AβP25-35 on BK channels in rat cerebral cortical neurons by using inside-out patch clamp technique. The results showed that: (1) the channel currents recorded in present study possess the characteristics of single BK channel: the average conductance of BK channels was 1 13 ?18.64 p.S (n = 7) in asymmetrical ([K+]i / [K+]0= 140/5 mmol/L )or 201 ?32.65 pS (n = 5) in symmetrical ([K+]j / [Kt]0= 140/140 mmol/L) K+ solution, respectively; and the reversal potential was -57.3 ?7.62 mV in asymmetrical K+ solution, which is closed to the K+ equilibrium potential; the open probability of these channels increased with the increase of [Ca2+]i and membrane depolarization, while the AβPlication of 140 mmol/L CsCl or 20 mmol/L TEA in bath solution completely blocked the activities of them; (2) After AβPlication of AβP31-35 (5 u,mol/L) to bath solution, the mean P0 and open frequency of BK channels decreased by 93.39% ?.72% (n = 17, p<0.01) and 54.84%?4.67% (n = 11, p<0.01), respectively;the mean open time of BK channels decreased by 54.67%?4.61 %, from being 7.51+5.68 ms to 2.71+3.43 ms (n = 10, p<0.01), while the mean current amplitude of BK channel was not significantly affected by the AβPlication of AβP31-35. (3) As in the case of AJ3P31-35, AβPlication of AβP25-35 (5umol/L) also induced a decrease of 86.91%?6.61% (n =7, p<0.01) in mean P0 of BK channel; the mean open time decreased by 54.67% ?4.61%, from being 4.59+1.55 ms to 2.74+2.08 ms (n = 6, p<0.01). (4) The suppression of AβP31-35 and AβP25-35 on BK channels was reversible in most of membrane patches.Our present results suggest that BK channels in cortical neurons might be one of the targeting sites of AβP in AD brain; the suppression of BK channel activities might contribute to the neurotoxicities inducedPart II A0P31-35 and A0P25-35 suppress IA channel in rat hippocampal CA1 neuronsHere, we investigated the acute electrical responses of rat hippocampal neurons to AβPlication of AβP31-35 and AβP25-35 by using whole-cell voltage clamp technique. The results showed that: (1) after AβPli...