Identification of the gate structure of ion channels

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In 1998 the crystal structure of the potassium channel was elucidated by MacKinnon's group and the structural parts of the gate was directly demonstrated. Based on this structural information some mutations were introduced into two kinds of potassium channels (Kir2.1, KAT1). For the inwardly rectifying potassium channels (Kir2.1) three charged residues exist in the loop between the pore helix and the inner helix. The negatively charged residues (AspィイD1152ィエD1, GluィイD1153ィエD1) were neutralized. We developed a method to evaluate the surface potential from the shift of the chord conductance-voltage curves. It was found that GluィイD1153ィエD1 modified the affinity of MgィイD12+ィエD1 ion and reduced the surface potential, thus decreasing the single channel conductance. When the concentration of external MgィイD12+ィエD1 was increased the open channel blocking appeared. From the kinetic analyses of the single channel recordings under MgィイD12+ィエD1 blocking condition it was suggested that MgィイD12+ィエD1 ion in the blocked state inhibited the closure of the gate. In the KAT1 channel a mutation in the C-terminal end of the pore helix (T256) altered the activation kinetics upon hyperpolarization. It was revealed that the transition rate between the closed state and the open state was susceptible to the mutation.