European Journal of Neuroscience, Vol. 11, pp. 1105–1108, 1999 European Neuroscience Asso.pdf

European Journal of Neuroscience, Vol. 11, pp. 1105–1108, 1999 © European Neuroscience Association SHORT COMMUNICATION Ca2 activation of hSlo K channel is suppressed by N-terminal GFP tag Elisabeth Meyer and Peter Fromherz Department of Membrane and Neurophysics, Max-Planck-Institute for Biochemistry, D-82152 Martinsried/Munchen, Germany ¨ Keywords: gating, green fluorescent protein, ion channel Summary The human slow poke (hSlo) K channel was tagged with GFP (green fluorescent protein) at the N-terminus of its α-subunit. The fusion protein was expressed transiently in HEK293 cells; it formed functional voltage-gated channels as shown by whole cell patch-clamp measurements. However, the tag lowered the voltage dependence of gating and it suppressed the typical left-shift of gating by intracellular binding of Ca2. The location of the GFP-tagged N-terminus was confirmed to be on the extracellular side by application of a monoclonal antibody to nonpermeabilized cells. Structural interpretations of the effects are discussed. Introduction Green fluorescent protein (GFP) has been used as a tag for the (Boehringer Mannheim, Germany). As a reporter protein we used N- and C-termini of proteins (Wang & Hazelrigg, 1994). Despite enhanced GFP (EGFP). pEGFP-C1 (Clontech, Palo Alto, CA, USA) the large size of GFP (238 amino acids), fusion proteins indicated was transformed into a dam– E. coli host (NEB, Schwalbach, Ger- the functional properties of the fusion partners. For example, an many), restricted with KpnI and Xba I and dephosphorylated. hSlo NMDA channel heteromer composed of an AR1 GFP-fusion and was ligated to the C-terminus of pEGFP.