Functional properties of a truncated recombinant GIRK5 potassium channel.
Xenopus laevis oocytes codify a G-protein-activated inward rectifier potassium channel (GIRK5 or Kir3.5). Coinjection of other GIRKs, the muscarinic m2 receptor, or Gbetagamma protein cRNAs is required to observe functional GIRKx-GIRK5 heteromultimers in oocytes. Studies with GIRK2 isoforms have shown that the size of the amino or carboxyl terminus plays a crucial role on giving functional K(+) channels. In this work we studied the properties of a GIRK5 with 25 amino acids deleted toward its amino-terminal domain. Injection of GIRK5-Delta25 cRNA alone displayed large basal and transient inward rectifying currents in oocytes. The instantaneous currents reached a stationary level after a long duration voltage pulse (10 s). For this relaxation, fast (tau(1)) and slow (tau(2)) time constants were estimated at different voltages. Recovery from inactivation followed a monoexponential function (tau=0.95+/-0.07 s). By contrast with other inward rectifier channels, blockade of GIRK5-Delta25 by extracellular Ba(2+) was voltage-independent (K(d)=102+/-2 microM), suggesting the presence of a Ba(2+) site at the external channel vestibule. To confirm this hypothesis, the Ba(2+) sensitivity of two charged mutants GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) at each of the external loops was determined. GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) showed a 100-fold and 2-fold higher affinity to Ba(2+), respectively, supporting the existence of this Ba(2+) binding site.
Salvador
C
C
Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F., México.
Martinez
M
M
Mora
S I
SI
Egido
W
W
Farias
J M
JM
Gamba
G
G
Escobar
L I
LI
D000818:Animals; D001464:Barium / Q000737:chemistry / Q000378:metabolism; D017609:Barium Compounds / Q000494:pharmacology; D001665:Binding Sites; D002413:Cations, Divalent; D002712:Chlorides / Q000494:pharmacology; D004594:Electrophysiology; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D055672:Static Electricity; D014162:Transfection; D014982:Xenopus laevis
D017609:Barium Compounds; D002413:Cations, Divalent; D002712:Chlorides; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D011994:Recombinant Proteins; C024986:barium chloride; D001464:Barium