Real-time detection of the surface delivery of newly synthesized membrane proteins.
Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.
Andreose
J S
JS
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Fumagalli
G
G
Sigworth
F J
FJ
Caplan
M J
MJ
D000818:Animals; D002038:Bungarotoxins / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D004594:Electrophysiology / Q000379:methods; D005583:Fourier Analysis; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007473:Ion Channels / Q000096:biosynthesis / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D051379:Mice; D018482:Muscle, Skeletal; D011499:Protein Processing, Post-Translational; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000502:physiology*; D013997:Time Factors
D002038:Bungarotoxins; D007473:Ion Channels; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate