Tagging with Green Fluorescent Protein Reveals a Distinct Subcellular Distribution of L-Type and Non-L-Type Ca2+Channels Expressed in Dysgenic Myotubes

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-02, Vol.95 (4), p.1903-1908
• Main Authors: Grabner, Manfred, Dirksen, Robert T., Beam, Kurt G.
• Format: Article
• Language: English
• Subjects: Albs; Animals; Biochemistry; Biological Sciences; Calcium; Calcium - metabolism; Calcium Channels - chemistry; Calcium Channels - metabolism; Calcium Channels, L-Type; Cell Compartmentation; Cell Membrane - metabolism; Cell membranes; Cells, Cultured; Cellular biology; Complementary DNA; Electric current; Fluorescence; Green Fluorescent Proteins; Ion Channel Gating; Luminescent Proteins; Mice; Microscopy; Microscopy, Confocal; Muscle Contraction; Muscle fibers; Muscles - metabolism; Muscles - ultrastructure; Myocardium - metabolism; Neurons - metabolism; Physiology; Proteins; Recombinant Fusion Proteins; Rodents
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.4.1903

Expression of cardiac L-type Ca2+channels in dysgenic myotubes results in large Ca2+currents and electrically evoked contractions resulting from Ca2+-entry dependent release of Ca2+from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca2+channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca2+currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca2+channels in close apposition to sarcoplasmic reticulum Ca2+release channels. We tagged the N termini of different α1subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged α1subunit exhibited Ca2+channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-α1Sand GFP-α1Cin dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-α1Aand GFP-α1Bfailed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-α1Sand GFP-α1Cwere present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-α1Aand GFP-α1Bwere not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.