Plasma membrane protein clusters appear in CFTB-expressing Xenopus laevis oocytes after cAMP stimulation

Membrane trafficking of the cystic fibrosis transmembrane conductance regul ator (CFTR) is supposed to be an important mechanism controlled by the intr acellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to vis ualize protein insertion we applied atomic force microscopy (AFM) to inside -out oriented plasma membrane patches of CFTR-expressing Xenopus laevis ooc ytes before and after cAMP-stimulation, In a first step, oocytes injected w ith CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. W ater-injected oocytes served as controls. Then, plasma membrane patches wer e excised, placed (inside out) on glass and scanned by AFM. Before cAMP-sti mulation plasma membranes of both water-injected and CFTR-expressing oocyte s contained about 200 proteins per mum(2). Molecular protein masses were es timated from molecular volumes measured by AFM. Before cAMP-stimulation, pr otein distribution showed a peak value of 11 nm protein height correspondin g to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBM X) plasma membrane protein density increased in water-injected oocytes to 7 00 proteins per mum(2) while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 300 proteins per mum(2) while protein distribution e xhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could repr esent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quan tified protein distribution with AFM at molecular level. We propose that CF TR causes clustering of plasma membrane proteins.