Electrostatic domino effect in the Shaker K channel turret

Voltage-gated K channels are regulated by extracellular divalent cations such as Mg2+ and Sr2+ either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl2 shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at similar to- 20mV and Kv2.1 channel open at; 15 mV. The aim of this study was to identify critical residues for this Mg2+ - induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg2+- shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425>419>451. Charging 425 and 419 not only affect the Mg2+- induced G(V) shift with 5 - 6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel's working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4-thus, an electrostatic domino effect.