Existence of a Ca2+-dependent K+ channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding

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

“…One class of channel, which has been carefully analyzed using a patch-clamp technique (3,17), is inhibited by tetraethylammonium ions and is insensitive to apamin. The other class of channels is blocked by apamin and is resistant to tetraethylammonium (10).…”

mentioning

confidence: 99%

The all-or-none role of innervation in expression of apamin receptor and of apamin-sensitive Ca2+-activated K+ channel in mammalian skeletal muscle.

Schmid-Antomarchi¹,

Renaud²,

Romey³

et al. 1985

“…Since we now find a G protein in the PSD, which interacts with calmodulin, we propose that the depolarization-induced reduction of the G-protein ADP-ribosylation (74) is caused by the movement of calmodulin from the kinase in the PSD to the G protein in the PSD upon depolarization. Thus, it could be that calmodulin links the G protein to the voltage-dependent Ca2+ channel, resulting in a structural change in the channel protein as shown by the change in nitrendipine binding (1,2), and in a change in the G protein as shown by a change in ADP-ribosylation (this paper). This could explain the modification by calmodulin of the binding of the Ca2+-channel blocker nitrendipine to both SM and PSD fractions (1,2) and the modification by calmodulin of the ADP-ribosylation of G proteins in the SM and PSD fractions.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it could be that calmodulin links the G protein to the voltage-dependent Ca2+ channel, resulting in a structural change in the channel protein as shown by the change in nitrendipine binding (1,2), and in a change in the G protein as shown by a change in ADP-ribosylation (this paper). This could explain the modification by calmodulin of the binding of the Ca2+-channel blocker nitrendipine to both SM and PSD fractions (1,2) and the modification by calmodulin of the ADP-ribosylation of G proteins in the SM and PSD fractions. Also, electrophysiological experiments with neuronal cells showed that the dihydropyridine analogues did directly interact with a G protein to change the ratios of Ca2+ channels in the active and inactive states (7,12,16), and chronic membrane depolarization of neuroblastoma cells increases the levels of the a subunit of Go (74).…”

Section: Discussionmentioning

confidence: 99%

Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin.

Nigám

Ledoux

et al. 1992

We have examined the isolated postsynaptic density (PSD) Pertussis Toxin-Activated ADP-Ribosylation. Pertussis toxin-activated ADP-ribosylation of SM and PSD proteins was performed as described using [32P]NADP (35). The samples were processed by SDS/PAGE and autoradiography (36), with the polyacrylamide gel being a 7.5-15% linear gradient in all cases. MATERIALS AND METHODS Materials. Guanosine 5'-[-[35S]thio]triphosphate (GTP[-35S]) (1400Immunochemical Characterization of the G Proteins. To identify the G proteins in the SM and PSD fractions, the subcellular fractions (60 ,ug each) were subjected to SDS/ PAGE and the resolved proteins in the gel were transferred electrophoretically to Immobilon (Millipore) paper as described (37). The Western blot blots were probed with anti-Ga antibodies followed by 125I-labeled protein A (38). 8686The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.