Pore mutations alter closing and opening kinetics in Shaker K+ channels

We have studied the effects of mutations of amino acids in the pore (positions 447 and 449) and the elevation of extracellular [K + ] on the closing and opening kinetics of Shaker B K + channels transiently expressed in Chinese hamster ovary (CHO) cells. Mutant D447E had closing and C-type inactivation kinetics which were faster than the wild-type channel. These processes were slowed by increasing extracellular [K + ] and in these conditions the channels exhibited linear instantaneous current-voltage relationships. Thus, the mutation seems to produce uniform decrease of occupancy by K + in sites along the channel pore where the cation competes with closing and C-type inactivation. In other mutants also showing K + -dependent fast C-type inactivation, closing was found to be slower than in the wild-type channel and insensitive to variations in external [K + ]. These characteristics were particularly apparent in mutant T449K which even in high [K + ] has a non-linear instantaneous current-voltage relationship with marked saturation of the inward current recorded at negative membrane potentials. Hence, in this channel type occupation by K + of the pore appears to be non-uniform with low occupancy of sites near the outer entrance and saturation of the sites accessible from the internal solution. The results show that channel closing is influenced by changes in the pore structure leading to alterations in the occupation of the channels by permeant cations. The differential effects of pore mutations and high external [K + ] on closing and C-type inactivation indicate that the respective gates are associated with separate domains of the molecule. Point mutations in the pore sequence can also lead to modifications in channel opening. In general, channels with fast C-type inactivation also show a fast rising phase of activation. However, these effects appear not to be due to primary modifications of the activation process but to arise from the coupling of activation and C-type inactivation. These data, demonstrating that the pore structure influences most of the gating parameters of K + channels, give further insight into the mechanisms underlying the modulation of K + channel function by changes in the ionic composition in the extracellular milieu.