Key result
N-terminal GFP tagging of the hSlo K+ channel lowered the voltage dependence of gating and suppressed the typical left-shift of gating by intracellular binding of Ca2+.
Tagging the hSlo K+ channel with GFP at the N-terminus alters its gating properties and suppresses Ca2+ activation, which must be considered when using this fusion protein in experimental models.
Caution warranted with GFP-tagged hSlo constructs in experiments; leaves open fidelity to native channel gating in physiological models.
The human slow poke (hSlo) K+ channel was tagged with GFP (green fluorescent protein) at the N-terminus of its alpha-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.
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Meyer et al. (1999) studied this question. N-terminal GFP tag on hSlo K+ channel was evaluated on Voltage dependence of gating and Ca2+ activation. N-terminal GFP tagging of the hSlo K+ channel lowered the voltage dependence of gating and suppressed the typical left-shift of gating by intracellular binding of Ca2+.
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