The human slow poke (hSlo) K+ channel was tagged with GFP (green fluorescent protein) at the N-terminus of its a-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 applying a monoclonal antibody to nonpermeabilized cells. Structural interpretations of the effects are discussed.
Figure: Calcium activated voltage-dependent gating of hSlo K+ channel. Normalized macroscopic conductance G/GMAX versus membrane potential for the untagged channel (open symbols) from right to left with 1 µM (diamonds), 30 µM (squares), 75 µM (circles) and 100 µM (triangles) intracellular Ca2+, and of the GFP-hSlo construct (full symbols) in 1 µM (diamonds) and 100 µM Ca2+ (triangles). The data are fitted with a voltage-dependent gating equilibrium.
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