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Residues beyond the selectivity filter of the K+ channel kir2.1 regulate permeation and block by external Rb+ and Cs+
G A Thompson1, M L Leyland, I Ashmole
1Ion Channel Group, Department of Cell Physiology and Pharmacology and Department of Chemistry, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
Abstract:
1. Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage-dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2. A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore-forming P-region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. 3. An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore-lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non-polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4. We tested the hypothesis that Asp172 - a residue implicated in channel gating by polyamines - formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. 5. We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild-type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.
Insights
Potassium channel (Kir2.1) mutations reveal Thr141 and Ser165 as key binding sites for blocking cations, influencing ion flow beyond the main selectivity filter.
Area of Science:
- Molecular Biology
- Biophysics
- Ion Channel Function
Background:
- Inward rectifier potassium channels (Kir2.1) are crucial for regulating membrane potential.
- Voltage-dependent block by ions like Rb+ and Cs+ is a characteristic feature of these channels.
- Understanding the molecular basis of ion block is essential for deciphering channel function.
Purpose of the Study:
- To investigate the role of specific residues, Ser165 and Thr141, in the pore of Kir2.1 channels.
- To elucidate the mechanism of voltage-dependent block by monovalent cations (Rb+, Cs+).
- To determine the contribution of Ser165 and Thr141 to ion permeation and blockage.
Main Methods:
- Site-directed mutagenesis of Kir2.1 channel residues (Ser165, Thr141, Asp172).
- Electrophysiological recordings (voltage-clamp) to measure ion currents (Rb+, Cs+).
- Homology modeling based on KcsA channel structure to predict pore architecture.
Main Results:
- Mutation S165L abolished Rb+ block and reduced Cs+ affinity, indicating Ser165's role in blockage.
- Mutations at Thr141 and Ser165 did not support a hydrogen bond model for blockage.
- Ser165 was identified as a pore-lining residue affecting ion blockage; Thr141 also contributes to cation binding sites.
Conclusions:
- Thr141 and Ser165 are likely binding sites for monovalent blocking cations in Kir2.1 channels.
- These residues are located beyond the primary selectivity filter, requiring traversing for ion blockage.
- The findings provide insights into the structural determinants of ion permeation and voltage-dependent block in Kir channels.