Functional properties of a truncated recombinant GIRK5 potassium channel

Abstract

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 Gβγ 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-Δ25 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 (τ 1) and slow (τ 2) time constants were estimated at different voltages. Recovery from inactivation followed a monoexponential function (τ=0.95±0.07 s). By contrast with other inward rectifier channels, blockade of GIRK5-Δ25 by extracellular Ba 2+ was voltage-independent ( K d=102±2 μM), 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-Δ25(N129E) and GIRK5-Δ25(K157E) at each of the external loops was determined. GIRK5-Δ25(N129E) and GIRK5-Δ25(K157E) showed a 100-fold and 2-fold higher affinity to Ba 2+, respectively, supporting the existence of this Ba 2+ binding site.