Differential localization of voltage-dependent calcium channel alpha1 subunits at the human and rat neuromuscular junction

Abstract

Neurotransmitter release is regulated by voltage-dependent calcium channels (VDCCs) at synapses throughout the nervous system. At the neuromuscular junction (NMJ) electrophysiological and pharmacological studies have identified a major role for P- and/or Q-type VDCCs in controlling acetylcholine release from the nerve terminal. Additional studies have suggested that N-type channels may be involved in neuromuscular transmission. VDCCs consist of pore-forming alpha1 and regulatory beta subunits. In this report, using fluorescence immunocytochemistry, we provide evidence that immunoreactivity to alpha1A, alpha1B, and alpha1E subunits is present at both rat and human adult NMJs. Using control and denervated rat preparations, we have been able to establish that the subunit thought to correspond to P/Q-type channels, alpha1A, is localized presynaptically in discrete puncta that may represent motor nerve terminals. We also demonstrate for the first time that alpha1A and alpha1B (which corresponds to N-type channels) may be localized in axon-associated Schwann cells and, further, that the alpha1B subunit may be present in perisynaptic Schwann cells. In addition, the alpha1E subunit (which may correspond to R/T-type channels) seems to be localized postsynaptically in the muscle fiber membrane and concentrated at the NMJ. The possibility that all three VDCCs at the NMJ are potential targets for circulating autoantibodies in amyotrophic lateral sclerosis is discussed.

Fig. 1.

Distribution of α_1A(A), α_1B (B), α_1E (C), neurofilament (D), S100 (E), and β-spectrin (F) at the human NMJ in transverse sections of gastrocnemius muscle. Sections were dual-labeled with FITC-conjugated DBA to identify the NMJ (left-hand images) and primary antibodies to the above proteins, followed by rhodamine-conjugated secondary antibodies (right-hand images). α_1A-ir (A) and α_1B-ir (B) were localized only at the NMJ. The α_1E antibody (C) labeled the NMJ and around the outside of the muscle fiber. Scale bar, 30 μm.

Fig. 2.

Distribution of α_1A(A, G), α_1B(B, H), α_1E(C, I), synaptophysin (D, J), S100 (E,K), and β-spectrin (F,L) at the rat NMJ in transverse sections from control (A–F) and denervated (G–L) soleus muscle. Sections were dual-labeled with BgTX to label postsynaptic acetylcholine receptors (left-hand images) and antibodies to the above proteins (right-hand images). In control sections (A–F) α_1A-ir (A), α_1B-ir (B), and α_1E-ir (C) were localized at the NMJ. In addition, α_1E-ir (C) was localized around the outside of the muscle fiber. In denervated sections (G–L) no labeling was observed with the α_1A antibody (G), whereas α_1B-ir (H) and α_1E-ir (I) looked similar to that in control sections. Scale bar, 30 μm.

Fig. 3.

Distribution of α_1A(A, H), α_1B (B, I), α_1E (C, J), neurofilament (D, K), synaptophysin (E, L), S100 (F, M), and β-spectrin (G, N) at the rat NMJ in teased muscle fibers from control (A–G) and denervated (H–N) soleus muscle. Teased fibers were dual-labeled with BgTX (left-hand images) and the above antibodies (right-hand images). In control teased fibers (A–G), α_1A exhibited punctate labeling at the NMJ and also labeled processes leading into each NMJ. The α_1B antibody (B) labeled the entire surface area of the NMJ and labeled processes leading into the NMJ. Labeling with the α_1E antibody was concentrated at the NMJ (C). In denervated teased fibers (H–N) no labeling of the NMJ could be detected with the α_1A antibody (H). In contrast, α_1B-ir (I) and α_1E-ir (J) were similar in denervated and control teased fibers. Scale bar, 30 μm.

Fig. 4.

Pseudocolored confocal microscope images of α_1A-ir (A, A′), α_1B-ir (B, B′), and α_1E-ir (C, C′) in control rat teased muscle fibers. Preparations were dual-labeled with BgTX (green) and α₁ subunit antibodies (red). Regions of colocalization are shown inyellow/orange. Low-magnification images (20× objective) reveal that α_1A-ir (A) and α_1B-ir (B) are present in preterminal processes, whereas α_1E-ir (C) is not. Higher-magnification images (40× objective) of single NMJs show the degree of colocalization of α₁-ir with BgTX labeling. The three subunit antibodies exhibited clearly different patterns of labeling. The α_1Aantibody (A′) exhibited a punctate labeling pattern that lies within the area demarcated by BgTX binding. In contrast, the α_1B (B′) antibody labeled the entire surface area of the NMJ, with labeling extending beyond the boundaries demarcated by BgTX binding, whereas α_1E-ir (C′) colocalized more precisely with BgTX binding. Scale bars: for A–C, 25 μm; for A′–C′, 20 μm.

Fig. 5.

Distribution of S100 (A), α_1A (B), α_1B(C), and α_1E(D) in rat sciatic nerve sections. The S100 antibody (A) intensely stained a double-ring structure, consistent with labeling of the outer and inner membrane of axon-associated Schwann cells. A similar but weaker pattern of labeling was observed with α_1A (B) and α_1B (C) antibodies, whereas α_1E-ir (D) was negligible in sciatic nerve sections. Scale bar, 15 μm.