Coupling of K(+)-gating and permeation with Ca2+ block in the Ca2(+)-activated K+ channel in Chara australis

The patch-clamp technique is used here to investigate the kinetics of Ca2+ block in single high-conductance Ca2(+)-activated K+ channels. These channels are detected in the membrane surrounding cytoplasmic drops from Chara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (-300 to 200 mV). Ca2+ on either side of the channel reduces its K+ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca2(+)-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca2+ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca2+ indicates that (i) external Ca2+ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca2+ or K+, and (iii) the kinetics of Ca2+ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca2+ indicates that more than one Ca2+ is involved.