Monitoring Expression and Clustering of the Ionotropic 5HT3Receptor in Plasma Membranes of Live Biological Cells

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Abstract
The ionotropic 5HT3 receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concentration of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was observed continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT3 receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently those receptors expressed later by another, spectrally distinguishable, high-affinity fluorescent ligand. Having reached a critical cell surface concentration of ∼3000 receptors/μm2, the receptors started to aggregate in patches with a 4-fold increased surface concentration. The clusters were constantly delivered from a pool of freshly expressed receptors isotropically distributed within the basolateral region of the cell membrane. From there, they migrated to and accumulated on the apical cell surface ∼9 h after transfection. Individual clusters grew until they reached a critical size of 1−2 μm when they merged to form with 3−5 μm large macroclusters. Clustered receptors were immobile on the minute time scale but always coexisted with monomeric receptors in the regions surrounding the clusters as revealed by fluorescence correlation spectroscopy. Because the receptor density of 12 000 receptors/μm2 in the patches is as high as that found in two-dimensional crystals of certain membrane proteins, such patches might be a proper source for direct crystallization of membrane proteins without prior purification.