1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix of the murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.
The cardiac muscarinic receptor‐K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell‐attached configuration of the patch clamp technique.
In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity as a result of desensitization, similar to that in atrial cells.
Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by ≈39 %) by transfecting with a mutant receptor kinase with little kinase activity.
These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation‐dependent and ‐independent mechanisms.
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