High K⁺ induces multiple amphiphysin I (Amphi. I) cleavages by calpain in mouse hippocampal slices. (A) Western blotting analysis of hippocampal slices stimulated with L-glutamate or high K⁺ and then allowed to rest for the indicated times. Probing with anti-amphiphysin I antibody recognizing the N terminus demonstrated the presence of three truncated forms of amphiphysin I. Arrow, fourth truncated form of amphiphysin I. (B) Time-dependent cleavages of amphiphysin I and α-spectrin after high K⁺ stimulation of hippocampal slices for the indicated times. The lowest panel shows the quantitative analysis of the expression changes of FL amphiphysin I and α-spectrin after high K⁺ stimulation. The expression levels of both proteins of the control (time=0 min) were set at 1. (C) Subcellular fractionation of μ- and m-calpain from cerebral cortex as shown by probing with anti-μ- and m-calpain antibodies. Anti-synaptotagmin antibodies were used to monitor synaptosomal fractions. H, total homogenate; P2, crude synaptosomal pellet; LP2, membrane fraction of purified synaptosomes; LS2, cytosol fraction of purified synaptosomes. (D) Anti-amphiphysin I immunoblotting showing that calpain inhibitors, ALLM (AM) or ALLN (AN), blocked high K⁺-induced amphiphysin I cleavage. (E) Anti-amphiphysin I immunoblotting showing the in vitro cleavage of recombinant amphiphysin I (0.5 μg/μl) by various concentrations of recombinant m-calpain.

Interaction of amphiphysin I (Amphi. I) with liposomes and dynamin I (Dyn. I) inhibits the cleavage by calpain. (A) Morphological analysis of the interaction of amphiphysin I with liposomes and dynamin I at the electron microscopic level. Amphiphysin I recruits dynamin I onto liposomes, forming collars on tubulated liposomes. Bars in upper panels, 2 μm, bar in lower panel, 100 nm. (B) Effects of the interaction of amphiphysin I with liposomes and dynamin I on its cleavage by calpain are shown by anti-amphiphysin I immunoblotting of liposome and liposome-free fractions of the incubation mixture. (C) Anti-dynamin I immunoblotting shows that dynamin I was not cleaved by calpain in the presence or absence of either liposomes or amphiphysin I. (D) The effect of liposomes on calpain activity was examined by incubating purified calcineurin in the presence or absence of liposomes. Calcineurin cleavage by calpain was examined by Western blotting with anti-calcineurin antibodies. (E) Comparison of calpain-dependent cleavage between WT amphiphysin I and its phospho-mimic mutant form in vitro. For the mutant, Cdk5 phosphorylation sites (S261, S272, S276, S285 and T310) of amphiphysin I were replaced with Asp (5D). Both types of recombinant amphiphysin I were incubated with 2 nM m-calpain for 30 min. (F) The effect of phosphorylation of amphiphysin I on its cleavage by calpain was examined in hippocampal slices treated with either FK506 (FK), an inhibitor of calcineurin, or roscovitine (Ros.), a Cdk5 inhibitor. Hippocampal slices were preincubated with and without 50 μM FK or 10 μM Ros. for 30 min at 30°C and were then treated with 80 mM KCl for 15 min. Homogenized slices were analyzed by immunoblotting with anti-amphiphysin I and anti-synaptotagmin antibodies.

Amphiphysin I cleavage by calpain inhibits excessive synaptic transmission in hippocampal slices and in vivo. (A) Anti-amphiphysin I immunoblot showing the effects of either no electrical stimulation (C), LFS (L, 0.03 Hz) or HFS (H, 10 Hz) on calpain-dependent amphiphysin I cleavage in mouse hippocampal slices, without or with ALLM preincubation. (B) ALLM inhibition of calpain had no effect on fEPSP slope during LFS. (n=7) (C) Effect of calpain inhibition (ALLM) on fEPSP depression induced by HFS. Control, n=8; ALLM, n=11. (D) Comparison of fEPSP slopes at 15 min after LFS and HFS in the ALLM-perfused slices and controls. ^*P<0.05. (E) PPF in hippocampal slices following LFS is reduced following HFS, an effect that is blocked by inhibiting calpain with ALLM. ALLM had no effect on PPF following LFS. The data represent the ratios of the second EPSP slopes versus the first fEPSP slopes separated by the given intervals. As a control, hippocampal slices were incubated with DMSO instead of ALLM. ^*P<0.05, ^**P<0.01 compared with Cont. n=10–15. (F) Anti-amphiphysin I immunoblot showing the ability of KA injections to induce calpain-dependent amphiphysin I cleavage in the hippocampus of FVB/NJ mice. Preincubation with ALLM inhibited the KA-induced cleavage of amphiphysin I. ^*Nonspecific band. (G) Preconditioning with low-dose KA prevented KA-induced development of seizures in the FVB/NJ mice and ALLM inhibited the preconditioning effect. Left panel, schematic time schedule of the animal experiments. Right panel, the maximum seizure score in mice of each group. Seizure scores were monitored for 3 h after KA (30 mg/kg) injection as described in ‘Methods'. ^*P<0.05.

Sites of cleavage of amphiphysin I by calpain. (A) Upper panel, schematic diagram of human amphiphysin I and the recognition sites of each anti-amphiphysin I antibody (recognizing the N terminus (I) or C terminus (II)). Lower panel, Western blotting showing the m-calpain (7 nM)-induced truncation products of recombinant amphiphysin I recognized by anti-amphiphysin I antibodies I and II. CBB staining showing amphiphysin I was mainly cleaved to three fragments corresponding to the truncated products detected by antibody I. (B) No truncated form of amphiphysin I was detected by antibody II in high K⁺-treated hippocampal slices. (C) Three major truncations of amphiphysin I. Upper panel, scheme of the cleavage sites by m-calpain identified by MS analysis. Lower panel, comparison of the molecular weights of amphiphysin I cleaved in vitro by m-calpain with those of the recombinant proteins of each truncation form identified by MS.

Amphiphysin I cleaved by calpain and a truncated form (1–392) are capable of tubulation with liposomes, but do not form ring structures with dynamin I (A: a–j), and inhibitory effect of calpain-dependent truncated form of amphiphysin I on transferrin uptake in Cos-7 cells (B) and FM 4–64 labeling in primary culture of hippocampal neurons (C). (A: a–d) The effect of truncation on the ability of amphiphysin I to form tubules with liposomes. As a chelator of Ca²⁺, EGTA inhibits calpain activity. (A: e–h) The effect of both forms of amphiphysin I on the formation of ring structures with dynamin I. (A: i, j) Comparison of helical collars on the tubules of FL and truncated amphiphysin I when incubated with dynamin I plus liposomes. Bars in A: d and h, 500 nm; bars in A: i and j, 100 nm. (B) Confocal laser scanning micrographs of: left panel, EGFP expression on COS-7 cells transfected with pIRES2-EGFP vector only (Vector), the vector containing wild-type amphiphysin I cDNA (WT) and the vector containing truncated amphiphysin I (1–392) cDNA (truncated form); middle panel, transferrin (Tf.) uptake in similarly transfected cells exposed to transferrin 24 h later; and, right panel, showing the overlay of EGFP and transferrin uptake. Transferrin uptake was inhibited in cells transfected with truncated amphiphysin I (arrows); bar, 10 μm. For quantification of the results, transferrin uptake was calculated as the mean fluorescence density for 315–660 cells under each condition and the mean values (±s.e.m.) were expressed as a percentage of normal transferrin uptake in untransfected cells. ^*P<0.01. (C) Fluorescence imaging micrographs of: left panel, EGFP expression on presynapses of neurons transfected with vector, WT amphiphysin I and the truncated form (amphi. 1–392); middle panel, subtracted images of FM 4–64 (loading minus unloading); right panel showing overlay of EGFP expression and FM 4–64 labeling. Bar, 10 μm. FM 4–64 labeling was inhibited in boutons transfected with truncated amphiphysin I (arrows). Right graph shows the results of quantitative analysis. FM 4–64 labeling was calculated as the integral fluorescence intensity for 47–55 boutons under each condition and the mean values (±s.e.m.) were expressed as a percentage of normal FM 4–64 labeling in untransfected boutons. ^*P<0.01.