Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex

Increasing evidence suggests that the postsynaptic density (PSD) plays a critical role in synaptic communication and plasticity. The major PSD protein (mPSDp), a calcium/calmodulin-dependent protein kinase, appears to be central to PSD function. The mPSDp has long been considered identical to the a subunit of the soluble calmodulin kinase II (a-CKII). However, mPSDp and a-CKII do differ in solubility and antigenicity, raising the possibility that the two proteins are distinct. To further derme the relationship between the two proteins, we purified the mPSDp to homogeneity from adult rat cerebral cortex and compared the proteins. In contrast to a-CKII, the purified mPSDp was insoluble in high concentrations of salt, various detergents, chelators of divalent cations, and the strong denaturant guanidine hydrochloride. The pI value of the mPSDp was 6.2, whereas that of a-CKII was 6.7-7.2. The purified mPSDp bound calmodulin in the presence of Ca2' and was autophosphorylated in a Ca2+/calmodulin-dependent manner. Polyclonal antiserum raised against mPSDp (anti-mPSDp) recognized purified mPSDp or mPSDp in synaptic membrane, indicating immunologic specificity among the synaptic proteins. Anti-mPSDp did not recognize a-CKII, whereas anti-a-CKII antibodies reacted only weakly with mPSDp, suggesting that the proteins are distinct but structurally similar. Moreover, sequence analysis of protease V8-digested polypeptides revealed that there was at least an 8-amino acid sequence, MLKVPNIS, that is not present in a-CKII. Finally, HPLC analysis of V8-digested fragments of mPSDp and a-CKII in parallel revealed dissimilar peptide patterns. Thus our observations suggest that mPSDp and a-CKII are similar but not identical. The unique physicochemical and structural properties of the mPSDp may provide insights into molecular mechanisms mediating synaptic plasticity.Numerous studies suggest that the postsynaptic density (PSD), a proteinaceous disc-shaped structure attached to the postsynaptic membrane of chemical synapses, plays a critical role in synaptic communication and plasticity (for review, see ref. 1). Remarkably, however, only few proteins in the PSD have been characterized biochemically. Cerebral cortical or hippocampal PSDs contain a predominant protein, the major PSD protein (mPSDp), that binds calmodulin (CaM) in the presence of Ca2+ (2) and appears to be an autophosphorylatable Ca2+/CaM-dependent protein kinase (3-5). These unique activities are of critical functional significance since Ca2+ influx is a key step in memory formation (6) and since protein phosphorylation is central to signal transduction (7-9). Indeed, by monitoring the mPSDp, we have previously found that impulse activity regulates synaptic molecular architecture in the developing and mature sympathetic superior cervical ganglion (10)(11)(12). Moreover, the synaptic molecule mPSDp is similarly regulated in the adult rat hippocampus (12).Despite its potential functional significance, the identity of the mPSDp and its relationship to the soluble ty...

Section: Resultsmentioning

confidence: 99%

“…However, CKII has been localized to the cytosol immunohistochemically (22). In direct contrast, the mPSDp, as part of PSD, is insoluble in high concentrations of salt, various detergents, and the denaturant guanidine hydrochloride (23)(24)(25)(26)(27)(28). Moreover, the mPSDp is relatively nonantigenic (K.W.…”

mentioning

confidence: 99%

On the identity of the major postsynaptic density protein.

Wu¹,

Huang²,

Adler³

et al. 1992

“…In the following experiments, SDS͞PAGE was carried out according to ref. 20. All experiments were performed three times.…”

Section: Materials [␥-mentioning

confidence: 99%

“…PSD samples (100 g each in 25 l) were incubated at room temperature for 10 min with 25 l of a buffer [90 mM triethanolamine, pH 7.8͞1.43 mM MgSO 4 ͞0.6 mM EDTA͞ 10.5 mM D,L-glyceraldehyde-3 phosphate (G3P)͞1 mM phenylmethylsulfonyl fluoride͞10 M leupeptin͞50 M microcystin-LR͞270 M ␤-NAD͞0.1 mM ADP) and 10 l of 1 mM 32 Pi. The mixtures were then processed for substrate phosphorylation in the absence or presence of Ca 2ϩ ͞CaM as described (20). For estimation of the amount of ATP formed endogenously, Ca 2ϩ ͞CaM-dependent phosphorylation of the PSD samples was performed in parallel by using 8.3 fmol, 16.6 fmol, and 33.2 fmol of [␥-32 P]ATP added exogenously.…”

Section: No-stimulated [Adenylate-32 P]nad Incorporation Into G3pd Inmentioning

confidence: 99%

The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide

Aoki

Elste

et al. 1997

Self Cite

109

following correction should be noted. Due to an editorial change at PNAS, the meaning of the last sentence on page 14046 was altered. The sentence originally read as follows: On the other hand, this structure does not reproduce the pharmacological properties of either P or Q channel exactly, as the ID 50 to sFTX and -Aga IVA for P-type channels is lower than for the ␣1A, ␣2␦, ␤Ib channels in HEK cells.Neurobiology. In the article "The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide" Kuo Wu, Chiye Aoki, Alice Elste, Adrienne A. Rogalski-Wilk, and Philip Siekevitz, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(13273)(13274)(13275)(13276)(13277)(13278), the quality of the reproduction of Fig. 2A was poor. The figure and its legend are shown below:Biochemistry. In the article "KSR stimulates Raf-1 activity in a kinase-independent manner" by Neil R. Michaud, Marc Therrien, Angela Cacace, Lisa C. Edsall, Sarah Spiegel, Gerald M. Rubin, and Deborah K. Morrison, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(12792)(12793)(12794)(12795)(12796), the following correction should be noted.Due to a printer's error, background was incorrectly added to (50 g) and 100 g each of the other fractions, in 100 l final volume, including whole homogenate (H), synaptosomes (Syn), synaptic plasma membranes (SPM), and crude synaptic vesicles (CSV), were incubated at 37°C for 15 min. NAD incorporation was performed in the absence (Ϫ) or presence (ϩ) of SNP as exogenous source of NO. The mixtures were subjected to SDS͞PAGE and then autoradiography. (B) Western blot analysis of the G3PD in the subcellular fractions. To confirm that the radioactive protein in the subcellular fractions was indeed G3PD, Western blot analysis was performed by using specific anti-G3PD antibodies as described.

“…Subcellular Fractionation. Synaptosomal and synaptic membrane (SM) fractions were prepared from pooled frozen SCGs according to published procedures (16). Binding of Calmodulin to the Ganglionic PSDp.…”

mentioning

confidence: 99%

Transsynaptic impulse activity regulates postsynaptic density molecules in developing and adult rat superior cervical ganglion.

Wu¹,

Black²

1988

Ganglionic postsynaptic density protein (PSDp) was used to monitor the influence of transsynaptic impulse activity on synaptic structure in the developing and adult rat superior cervical sympathetic ganglion (SCG). Since transsynaptic activity is known to regulate ontogeny of postsynaptic transmitter enzymes, we initially studied the developing ganglion. Denervation in neonates prevented normal development, decreasing calmodulin binding to the ganglionic PSDp by 71% after 4 weeks. During this period, denervation elicited only a 42% decrease in total protein of the synaptic membrane fraction, suggesting that innervation regulates development of various synaptic components differentially. Effects of denervation were extremely rapid, resulting in a 44% decrease in calmodulin binding within 1 day, consistent with regulation by a signaling process such as impulse activity. The effect of impulse activity was examined more directly in adults by treatment with the agents reserpine or phenoxybenzamine, which elicit reflex increases in sympathetic transmission. Administration of reserpine resulted in a progressive 90% increase in calmodulin binding to the PSDp over 4 weeks. Phenoxybenzamine also elicited an increase, mimicking the effects of reserpine. Neither agent altered total protein of the synaptic membrane fraction, suggesting that impulse activity regulates specific synaptic components. Finally, ganglionic denervation in adults decreased PSDp binding within 12 hr, consistent with acute effects of impulse reduction. Our results suggest that transsynaptic impulse activity plays an important role in regulation of specific molecular components of the synapse.