| Resumo: |
We have constructed a fusion protein between mSlo (a recombinant, high conductance, calcium-activated potassium channel or maxi-K), and GFP (green fluorescent protein). This construct represents a tag to not only monitor channel expression, but to locate the protein in living cells. The GFP was fused in frame to the carboxy-terminus of the mSlo core protein (mSlo-GFP fusion protein). Expression of this fusion protein in COS-7 cells resulted in robust fluorescence localized near the cell membrane. Fluorescing cells that were patch clamped exhibited whole cell currents with a direction consistent with potassium currents. Conversely, non-fluorescing cells showed no significant whole cell currents. Excised inside out patches revealed single channel currents and calculated conductances in the range of those expected for the maxi-K. The mSlo and mSlo-GFP channels reconstituted into lipid bilayers bound wild-type, recombinant CTX with high affinity and displayed a half-blocking concentration (K D) of 7.4 and 7.6 nM, respectively (at +30 mV in 150 mM equimolar KCl). This resulted in single channel evaluation of the functional inhibition of CTX on these clones. As newly constructed GFP chimeras emerge for the study of physiological processes in living organisms, this work provides another area of insight illuminated by GFP.
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