The positive charge at 6’K is not essential for activation of GIRK2 channels. (a) Model shows a single subunit of GIRK2 (PDB: 4KFM), highlighting the region involved in binding PIP₂. Four lysines form hydrogen bonds with the 4′ and 5′ phosphates of PIP₂. Amino acid sequences of the tether helix for mouse GIRK2 (Kir3.2) and chicken Kir2.2 are shown below with the proposed number system. Arrow indicates HBC. (b) Current trace for GIRK2*-5′C shows little effect of 5 µM carbachol (C), 100 mM ethanol (E), 100 mM MPD (M) or Ba²⁺ before or after exposure to 1 mM MTS-HE. Vh= −100 mV. (c) Current trace for GIRK2*-6′C shows large activation of basal current with 1 mM MTS-HE. Note the inhibition of MTS-HE–activated current with both Ba²⁺ and MPD. (d) Bar graph shows mean increase in basal current density (pA/pF) measured at −100 mV. (e) Current trace for GIRK2*-6′C shows large activation of basal current with 0.1 mM MTS-F. (f) Mean fold-induction of basal current with 0.1 mM MTS-HE, 0.1 mM MTS-F, and 0.1 mM MTS-Y for GIRK2*-6′C. Bars show mean ± SEM.