External K+ relieves the block but not the gating shift caused by Zn2+ in human Kv1.5 potassium channels

摘要

class="enumerated" style="list-style-type:decimal">We used the whole-cell recording technique to examine the effect of extracellular Zn²⁺ on macroscopic currents due to Kv1.5 channels expressed in the human embryonic kidney cell line HEK293.Fits of a Boltzmann function to tail current amplitudes showed that 1 mm Zn²⁺ shifted the half-activation voltage from -10.2 ± 0.4 to 21.1 ± 0.7 mV and the slope factor increased from 6.8 ± 0.4 to 9.4 ± 0.7 mV. The maximum conductance in 1 mm Zn²⁺ and with 3.5 mm K⁺o was 33 ± 7 % of the control value.In physiological saline the apparent KD for the Zn²⁺ block was 650 ± 24 μm and was voltage independent. A Hill coefficient of 1.0 ± 0.03 implied that block is mediated by the occupation of a single binding site.Increasing the external concentration of K⁺ ([K⁺]o) inhibited the block by Zn²⁺. Estimates of the apparent KD of the Zn²⁺ block in 0, 5 and 135 mm K⁺ were 69, 650 and 2100 μm, respectively. External Cs⁺ relieved the Zn²⁺ block but was less effective than K⁺. Changing [K⁺]o did not affect the Zn²⁺-induced gating shift.A model of allosteric inhibition fitted to the relationship between the block by Zn²⁺ and the block relief by external K⁺ gave KD estimates of ≈70 μm for Zn²⁺ and ≈500 μm for K⁺.We propose that the gating shift and the block caused by Zn²⁺ are mediated by two distinct sites and that the blocking site is located in the external mouth of the pore.