Differences in expression of acetylcholinesterase and collagen Q control the distribution and oligomerization of the collagen-tailed forms in fast and slow muscles

Abstract

The collagen-tailed forms of acetylcholinesterase (AChE) are accumulated at mammalian neuromuscular junctions. The A(4), A(8), and A(12) forms are expressed differently in the rat fast and slow muscles; the sternomastoid muscle contains essentially the A(12) form at end plates, whereas the soleus muscle also contains extrajunctional A(4) and A(8) forms. We show that collagen Q (ColQ) transcripts become exclusively junctional in the adult sternomastoid but remain uniformly expressed in the soleus. By coinjecting Xenopus oocytes with AChE(T) and ColQ mRNAs, we reproduced the muscle patterns of collagen-tailed forms. The soleus contains transcripts ColQ1 and ColQ1a, whereas the sternomastoid only contains ColQ1a. Collagen-tailed AChE represents the first evidence that synaptic components involved in cholinergic transmission may be differently regulated in fast and slow muscles.

Fig. 1.

Immunofluorescence localization of AChE and ColQ proteins in the sternomastoid (STM) and soleus (SOL) muscles of newborn (P3) and adult (Ad) rats. Muscle sections were doubly stained with antibodies to AChE and ColQ. At both stages of development, the proteins colocalize in clusters. Scale bar: adult (Ad) muscles, 10 μm; newborn (P3) muscles, 5 μm.

Fig. 2.

In situ hybridization of AChE and ColQ transcripts in whole-mount sternomastoid (STM) and soleus (SOL) muscles of newborn (P3) and adult rats. In newborn (P3) and adult (Ad) muscles, AChE mRNAs are concentrated in the synaptic zone. ColQ mRNAs are diffuse along the muscle fibers, except in adult STM. Scale bar: adult STM and soleus hybridized with the ColQ probe, 15 μm; adult soleus and STM hybridized with the AChE probe and P3 soleus and STM hybridized with the ColQ probe, 12 μm; P3 soleus and STM hybridized with the AChE probe, 6 μm.

Fig. 3.

Quantitative relationship between AChE and ColQ transcripts in fast and slow muscles. A, RNase protection assay analysis: 15 μg of total RNA from junctional (j) and extrajunctional (ej) domains of the soleus (SOL) and sternomastoid (STM) muscles was hybridized with a ColQ probe covering the PRAD exon and the first part of the collagen domain, an AChE probe covering part of the catalytic domain, and a GAPDH probe, used as standard. After digestion with RNase, the protected fragments were separated in nondenaturing polyacrylamide gels that were then dried and exposed for 2 weeks to an autoradiographic film. To obtain comparable intensities, the specific radioactivity of the GAPDH probe was $\frac{1}{160}$ of that of the ColQ and AChE probes. B, Histogram representation of the relative abundance of the different transcripts, normalized to A content and GAPDH.

Fig. 4.

The pattern of collagen-tailed A₁₂, A₈, and A₄ is determined by the quantity of AChE_T. Xenopusoocytes were injected with a fixed amount of ColQ1 mRNA (0.25 ng/oocyte) or with water (control), together with a low level of AChE_T mRNA (0.62 ng/oocyte) (A) or with a high level of AChE_T mRNA (2.5 ng/oocyte) (B). The activity is similar with or without ColQ and proportional to the amount of injected AChE mRNA. AChE oligomers were separated in 5–20% sucrose gradients containing salt and Brij 96, so that the G₁ and G₂ forms sediment at 2 and 3 S. The identification of the oligomers is deduced from their position in the gradient relative to internal sedimentation markers. Note that the level of the 13.7 S oligomer depends on the level of AChE_T expressed in the cell. The Xenopusoocytes did not produce any detectable level of tetrameric G₄ form when expressing AChE_T alone. A 10 S G₄ form appeared, however, when it was expressed with ColQ, indicating that this form was organized by the PRAD. These tetramers may either contain a complete ColQ subunit, which did not form a collagen trimer, or only its N-terminal fragment.

Fig. 5.

Alternative 5′ exons of the ColQ gene.A, Peptide sequences encoded by rat exon 1, exon 1a, and exon 2. The predicted signal peptide is shown in bold type. B, Differential expression of the alternative initiation site variants: 15 μg of RNA from soleus muscle (SOL) and heart ventricle (VEN) was analyzed by RPA with two probes, covering alternative exons 1 or 1a and 2. Exon 1 is the only variant found in ventricle, whereas exon 1a is the major variant in the soleus.

Fig. 6.

Quantitative relationship between ColQ1 and ColQ1a in fast and slow muscles. RPA analysis of the 5′ end of ColQ1a and ColQ1: 15 μg of RNA from whole 3 d muscles (P3) and from adult (Ad) muscles [extrajunctional domain of soleus (SOL) and junctional domain of sternomastoid muscle (STM)] were hybridized with probes covering parts of exon 1a or exon 1 and exon 2, together with a GAPDH probe, used as standard. After digestion with RNase, the protected fragments were analyzed as in Figure 3.

Fig. 7.

The pattern of collagen-tailed A₁₂, A₈, and A₄ forms is determined by the relative amount of ColQ1 and ColQ1a mRNAs.Xenopus oocytes were injected with a fixed amount of AChE_T mRNA (2 ng/oocyte) together with 0.3 (A, A′), 0.15 (B,B′), and 0.07 (C, C′) ng of ColQ1a mRNA (left: A,B, C) or ColQ1 mRNA (right: A′, B′,C′). We obtained similar patterns of molecular forms with approximately twofold to threefold more ColQ1 mRNA than ColQ1a mRNA. At appropriate concentrations, both transcripts could generate either A₁₂ or incomplete A forms.