Stanley C. Froehner

The postsynaptic 43k protein clusters muscle nicotinic acetylcholine receptors in xenopus oocytes

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

The blood-nerve barrier is rich in glucose transporter

SummaryThe glucose transporter of the facilitated diffusion type has been localized in sections of innervated rat diaphragm muscle and sciatic nerve by immunofluorescence, using affinity-purified antibodies against both the entire transporter and the carboxy-terminal peptide. In both tissues the transporter was very abundant in the perineurial sheath of cells surrounding the nerve fibres. The transporter also appeared to be abundant in the endoneurial blood vessels of the sciatic nerve. The identity of the antigen as the glucose transporter was established by extracting sciatic nerve with sodium dodecylsulphate and immunoblotting the extract. A single reactive polypeptide with the expected molecular weight of 55 000 was found. The high concentration of glucose transporter in the cells of the blood—nerve barrier presumably ensures an adequate supply of glucose to the nerve fibres.

Localization of the α1 and α2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads

Abstract We have studied the subcellular distribution of the α 1 and α 2 subunits of the dihydropyridine (DHP) receptor and ankyrin in rat skeletal muscle with immunofluorescence and immunogold labeling techniques. All three proteins were concentrated in the triad junction formed between the T-tubules and sarcoplasmic reticulum. The α 1 and α 2 subunits of the DHP receptor were colocalized in the junctional T-tubule membrane, supporting their proposed association in a functional complex and the possible participation of the α 2 subunit in excitation-contraction coupling. Ankyrin label in the triad showed a distribution different from that of the DHP receptor subunits. In addition, ankyrin was found in longitudinally oriented structures outside the triad. Thus, ankyrin might be involved in organizing the triad and in immobilizing integral membrane proteins in T-tubules and the sarcoplasmic reticulum.

The role of the postsynaptic cytoskeleton in AChR organization

Abstract ACh receptors (AChRs) occur at much higher concentrations immediately adjacent to the nerve terminal than elsewhere on the muscle membrane. The molecular mechanisms responsible for maintaining this distribution are largely unknown but are thought to involve other synaptic proteins. Of particular interest are those proteins that comprise the postsynaptic cytoskeleton. This article discusses the role that both general and synapse-specific cytoskeletal proteins may play in the postsynaptic organization of receptors.

The α1 and α2 polypeptides of the dihydropyridine-sensitive calcium channel differ in developmental expression and tissue distribution

Abstract The dihydropyridine (DHP) binding complex isolated from skeletal muscle contains two large proteins, α 1 and α 2 , and at least two smaller polypeptides. The α 1 subunit has a primary structure expected for ion channels and is a functional component of a DHP-sensitive, voltage-activated calcium channel. The functions of the α 2 protein and the smaller polypeptides are unknown. We prepared monoclonal antibodies to the α 1 and α 2 polypeptides and studied the developmental appearance and tissue distribution of these two proteins. In rat skeletal muscle, the levels of α 1 are quite low during the first 10 days after birth, then rise dramatically, and, by day 20, approach those found in adult muscle. In direct contrast, α 2 is present in substantial amounts in rat muscle at birth and increases only slightly during this same period of development. Furthermore, α 1 is detected only in skeletal muscle, whereas α 2 is present in a variety of tissues. These results provide evidence for the segregation of these two polypeptides and suggest that the function of α 2 is not limited to that associated with the DHP-sensitive calcium channel.

Expression of RNA transcripts for the postsynaptic 43 kDa protein in innervated and denervated rat skeletal muscle

A cDNA clone encoding the mouse muscle postsynaptic 43 kDa protein was isolated and sequenced. The amino acid sequence of this protein, which is closely associated with nicotinic acetylcholine receptors at Torpedo electrocyte and vertebrate skeletal muscle synapses, is very similar in different species. A cysteine‐rich region homologous to part of the regulatory domain of protein kinase C may be important in interactions of this protein with the lipid bilayer. RNA transcripts for the 43 kDa protein increase only 2–3‐fold after denervation of rat skeletal muscle, in sharp contrast to the α‐subunit of the muscle nicotinic receptor which increases more than 30‐fold. Thus, the expression of these two proteins is regulated by different mechanisms.

Immunochemical detection of proteins biotinylated on nitrocellulose replicas

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Application of the enzyme substrate, alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization and is inhibited by biotinylated bovine serum albumin or 10 nM biotin. The procedure permits detection of less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.

Clusters of 43-kDa protein are absent from genetic variants of C2 muscle cells with reduced acetylcholine receptor expression☆

Genetic variants of the C2 muscle cell line were used to investigate the relation between acetylcholine receptor (AChR) clustering and clustering of the 43-kDa protein. Two variants that express severely reduced amounts of the alpha subunit of the AChR and consequently lack AChR clusters were found also to lack clusters of the 43-kDa protein. The amount of 43-kDa protein in the variants measured by immunoassay was reduced to about one-third the levels found in wild-type cells. The beta subunit of the AChR was reduced to a similar extent. Northern blot analysis showed that neither the 43-kDa protein mRNA nor the beta subunit mRNA was reduced in the variants. Taken together, these results suggest that the amounts of beta subunit and 43-kDa protein may be regulated coordinately by a post-transcriptional mechanism.

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.

Staining of proteins on nitrocellulose replicas

Publisher Summary This chapter describes a chemically defined technique whereby amino groups of proteins are covalently derivatized with sulfosuccinimidobiotin after transfer to nitrocellulose paper. Depending on the sensitivity required, either of two techniques can be used to stain the proteins. The first method utilizes avidin or streptavidin conjugated to horseradish peroxidase. This procedure is rapid and detects less than 25 ng of protein in a single band. The second technique requires sequential incubations with streptavidin, rabbit anti-streptavidin polyclonal antibody, and goat anti-rabbit IgG conjugated to horseradish peroxidase. After electrophoretic transfer of the proteins to nitrocellulose membranes, the replicas are rinsed briefly in 0.1 M sodium bicarbonate and then soaked in the same buffer for five minutes. The replicas are then transferred to the same volume of bicarbonate buffer containing 10 μM sulfosuccinimidobiotin. Antibody activity to streptavidin is monitored with a solid-phase assay. Except for the washing steps, all volumes are 50 μl.