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Mutations in the putative pore-forming segment favor short-lived wild-type Kir2.1 pore conformations
Ruth A Schwalbe1, Charles S Wingo, Shen-Ling Xia
1Nephrology Section, Department of Veterans Affairs Medical Center, Division of Nephrology, Hypertension, and Transplantation, University of Florida, Gainesville, Florida 32610, USA. schwara@medicine.ufl.edu
Abstract:
We have characterized single and double mutations in the M1-M2 segment of an inwardly rectifying K(+) channel, Kir2.1, using the cell-attached configuration of the patch-clamp technique. These mutations generated novel N-glycosylation sites at positions 128, 140, 143, and 147. Previously, we showed that these mutants were glycosylated, functional, and at the cell surface, which indicated that the putative pore-forming segment, including the invariant G(Y/F)G sequence of K(+) channels, was extracellular [Schwalbe, R. A., Rudin, A., Xia, S.-L., and Wingo, C. S. (2002) J. Biol. Chem. 277, 24382-24389]. In this study, three conductance states, corresponding to the main open state and two subconductance states, were identified in WT Kir2.1 channels expressed in infected Sf9 cells. Kir2.1 channels with mutations in the M1-M2 linker had at least one distinguishable conductance state of WT channels. In addition, these mutations altered the transitions, duration, and frequency of the defined populations of permeating and nonpermeating states. Of note, S128N had permeation rates similar to those of WT Kir2.1, but the total duration of the lower subconductance state was 3-5 times longer. Mutations in the signature sequence, I143N/Y145T, produced channels with permeation rates similar to those of the main open state and lower subconductance state of WT Kir2.1; however, the frequencies of these states were substantially different. These results demonstrate a novel functional role of the M1-M2 segment in regulating the transitions of the Kir2.1 channel and therefore suggest that this segment is a critical structural determinant in adjustments of pore conformations. Additionally, our results indicate that these mutants are correctly folded and thus further substantiate that the M1-M2 segment, including the G(Y/F)G sequence, is topologically extracellular.
Insights
Mutations in the M1-M2 segment of Kir2.1 potassium channels alter channel gating and conductance states. This highlights the M1-M2 segment
Area of Science:
- Molecular biology
- Biophysics
- Ion channel physiology
Background:
- Inwardly rectifying potassium channels (Kir2.1) are crucial for cellular membrane potential.
- Previous studies indicated the pore-forming segment of Kir2.1, including the G(Y/F)G sequence, is extracellular.
- N-glycosylation sites were engineered into the M1-M2 segment of Kir2.1.
Purpose of the Study:
- To investigate the functional role of the M1-M2 segment in Kir2.1 channel gating.
- To characterize the conductance states and gating kinetics of Kir2.1 mutants.
- To further substantiate the extracellular topology of the M1-M2 segment.
Main Methods:
- Site-directed mutagenesis to introduce N-glycosylation sites in Kir2.1.
- Cell-attached patch-clamp electrophysiology to record channel activity.
- Analysis of single-channel currents to determine conductance states and kinetics.
Main Results:
- Wild-type Kir2.1 channels exhibit three conductance states: main open and two subconductance states.
- Mutations in the M1-M2 segment altered the transitions, duration, and frequency of these conductance states.
- Specific mutations (S128N, I143N/Y145T) modulated permeation rates and state occupancy, affecting channel gating.
Conclusions:
- The M1-M2 segment plays a critical role in regulating Kir2.1 channel gating and pore conformation.
- Mutations in this segment significantly impact channel kinetics and state transitions.
- The findings support the extracellular localization of the M1-M2 segment and the G(Y/F)G pore-forming sequence.