Abstract
In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations </=30 microM. In this paper, we further characterized the TEA modulation site and examined how varying K(+) occupancy of the pore influenced the interaction of K(+) with this site. In the presence of high internal and external [K(+)], TEA blocked 100% of current with an IC(50) of 1.9 +/- 0.2 mM. In the absence of a substitute permeating ion, such as Na(+), reducing access of K(+) to the pore resulted in a reduction of TEA efficacy, but produced little or no change in TEA potency (under conditions in which maximal block by TEA was just 32%, the IC(50) for block was 2.0 +/- 0.6 mM). The all-or-none nature of TEA block (channels were either completely sensitive or completely insensitive), indicated that one selectivity filter binding site must be occupied for TEA sensitivity, and that one selectivity filter binding site is not involved in modulating TEA sensitivity. At three different levels of K(+) occupancy, achieved by manipulating access of internal K(+) to the pore, elevation of external [K(+)] shifted channels from a TEA-insensitive to -sensitive state with an EC(50) of approximately 10 mM. Combined with previous results, these data demonstrate that the TEA modulation site has a high affinity for K(+) when only one K(+) is in the pore and a low affinity for K(+) when the pore is already occupied by K(+). These results indicate that ion-ion interactions occur at the selectivity filter. These results also suggest that the selectivity filter is the site of at least one low affinity modulatory effect of external K(+), and that the selectivity filter K(+) binding sites are not functionally interchangeable.
Highlights
Voltage-gated Kϩ channels are multi-ion pores, and, while conducting, can be occupied by three to four Kϩ (Hodgkin and Keynes, 1955; Neyton and Miller, 1988)
TEA sensitivity was reinstated at [Kϩ] as low as 30–100 M, and increased over the same [Kϩ] range as that required to block Naϩ current through the channel. This suggested that occupancy of a high affinity, selectivity filter binding site by Kϩ was involved in modulation of TEA sensitivity (Immke et al, 1999)
515 Immke and Korn from blocking the channel, (b) TEA efficacy is regulated by just one of the two selectivity filter cation binding sites, (c) the apparent affinity of Kϩ for the selectivity filter site that modulates TEA sensitivity differs depending on whether the other selectivity filter site is occupied by Kϩ, and (d) the apparent affinity of the outermost selectivity filter site, when the innermost site is occupied by Kϩ, is ف10 mM
Summary
Voltage-gated Kϩ channels are multi-ion pores, and, while conducting, can be occupied by three to four Kϩ (Hodgkin and Keynes, 1955; Neyton and Miller, 1988). The KcsA structure is remarkably consistent with a repulsion model of multi-ion permeation proposed for both Kϩ and Ca2ϩ channels (Neyton and Miller, 1988; Kuo and Hess, 1993) In this model, either of the two selectivity filter sites can bind Kϩ with high affinity if just a single cation is interacting with the selectivity filter. TEA sensitivity was reinstated at [Kϩ] as low as 30–100 M, and increased over the same [Kϩ] range as that required to block Naϩ current through the channel This suggested that occupancy of a high affinity, selectivity filter binding site by Kϩ was involved in modulation of TEA sensitivity (Immke et al, 1999). Our data indicate that ion–ion interactions occur at the selectivity filter, and that the selectivity filter is the site of at least one low affinity interaction of external Kϩ with the pore
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