1. The molecular basis of selectivity and gating were investigated in wild-type and mutant forms of the inward rectifier K+ channel Kir2.1 (IRK1). 2. Kir2.1 channels show characteristic time-dependent gating kinetics due to a reversible voltage-dependent channel block by cytoplasmic polyamines. Mutations at Asp 172 in the M2 domain revealed that a negative charge at this position is the main criterion of time-dependent gating kinetics in Kir2.1. 3. Kir channels are blocked by Cs+ and Rb+ in a voltage-dependent manner, characteristic of many Kir channels. Rb+ and Cs+ block in Kir2.1 was abolished by replacing Asp 172 by Asn, but was re-established by a change to Gln, narrowing the pore. However, blocking affinity was reduced by the mutation to Gln. 4. When Asp 172 was mutated to Glu, narrowing the pore but retaining the negative charge, block by both Cs+ and Rb+ was increased relative to wild-type. 5. Replacing Gly 168 in M2 by Ala was suggested to widen the pore at position 172. The effect of this mutation on Cs+ and Rb+ block was relatively small. 6. There appears to be a balance between charge and pore size in determining whether icons block or permeate. A major part of the selectivity of Kir2.1 is associated with Asp 172 in the M2 domain. This site also determines the time-dependent activation gating of the channel. 7. Mutation of Asp 172 to Ser were predicted to abolish both Cs+ and Rb+ block in Kir2.1, but blocking affinity was similar to WT. Therefore, other properties of the pore must contribute to Cs+ and Rb+ block at position 172 in addition to the electrostatic and steric effects identified in this study.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:696787 |
| Date | January 2000 |
| Creators | Abrams, Christopher John |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/29921 |
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