Dataset Information

Optical detection of rate-determining ion-modulated conformational changes of the ether-a-go-go K+ channel voltage sensor.

ABSTRACT: In voltage-dependent ether-à-go-go (eag) K+ channels, the process of activation is modulated by Mg2+ and other divalent cations, which bind to a site in the voltage sensor and slow channel opening. Previous analysis of eag ionic and gating currents indicated that Mg2+ has a much larger effect on ionic than gating current kinetics. From this, we hypothesized that ion binding modulates voltage sensor conformational changes that are poorly represented in gating current recordings. We have now tested this proposal by using a combined electrophysiological and optical approach. We find that a fluorescent probe attached near S4 in the voltage sensor reports on two phases of the activation process. One component of the optical signal corresponds to the main charge-moving conformational changes of the voltage sensor. This is the phase of activation that is well represented in gating current recordings. Another component of the optical signal reflects voltage sensor conformational changes that occur at more hyperpolarized potentials. These transitions, which are rate-determining for activation and highly modulated by Mg2+, have not been detected in gating current recordings. Our results demonstrate that the eag voltage sensor undergoes conformational changes that have gone undetected in electrical measurements. These transitions account for the time course of eag activation in the presence and absence of extracellular Mg2+.

SUBMITTER: Bannister JP

PROVIDER: S-EPMC1317915 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:In ether-à-go-go (eag) K(+) channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are approximately 4 A apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg(2+). In I287D+F324D, Mg(2+) significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg(2+) binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg(2+) shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg(2+) binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.

Dataset Information

Optical detection of rate-determining ion-modulated conformational changes of the ether-a-go-go K+ channel voltage sensor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets