Professor Stephen Tucker

Abstract:

A PCR-based cloning strategy was used to identify novel subunits of the two-transmembrane domain inward rectifier potassium channel family from rat brain, heart, and skeletal muscle. When expressed in Xenopus oocytes, two of these clones (Kir4.1 and Kir2.3) gave rise to inwardly rectifying potassium currents. Two-electrode voltage clamp commands to potentials negative to EK evoked inward potassium-selective currents which rapidly reached a peak amplitude and then relaxed to a steady-state level. Differences in the extent of current relaxation, the degree of rectification, and the voltage-dependent block by external cesium were detected. Two other members of this family (Kir5.1 and Kir3.4) did not produce macroscopic currents, when expressed by themselves, yet both subunits modified the currents when coexpressed with other specific members of the Kir family. Expression of chimeric subunits between Kir4.1 and either Kir5.1 or Kir3.4 suggested that the transmembrane domains determine the specificity of subunit heteropolymerization, while the C-terminal domains contribute to alterations in activation kinetics and rectification. Expression of covalently linked subunits demonstrated that the relative subunit positions, as well as stoichiometry, affect heteromeric channel activity.

Abstract:

Weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium-selective to a nonspecific cation-selective pore. In this study we confirm by cell-attached patch-clamp recording that the mutation produces a non-selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild-type Kir 3.2 allele, or other members of the Kir 3.0 subfamily. These results suggest that the weaver defect in Kir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild-type subunits of the Kir 3.0 subfamily.

Abstract:

Coexpression in Xenopus oocytes of the cloned cardiac inward rectifier subunits Kir 3.1 and Kir 3.4 results in G protein-stimulated channel activity closely resembling the muscarinic channel underlying the inwardly rectifying K+ current in atrial myocytes. To determine the stoichiometry and relative subunit positions within the channel, Kir 3.1 and Kir 3.4 were coexpressed in varying ratios with cloned G beta 1 gamma 2 subunits and also as tandemly linked tetramers with different relative subunit positions. The results reveal that the most efficient channel comprises two subunits of each type in an alternating array within the tetramer. To localize regions important for subunit coassembly and G protein sensitivity, chimeric subunits containing domains from either Kir 3.1, Kir 3.4, or the G protein-insensitive subunit Kir 4.1 were expressed. The results demonstrate that the transmembrane domains dictate the potentiation of the coassembled channels and that, although the NH4- or COOH-termini of both subunits alone can confer G protein sensitivity, both termini are required for maximal stimulation by G beta 1 gamma 2.

Abstract:

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co-injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co-expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4-5 or 4-5-4-5, but not when they were connected 4-4-5-5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4-5-4-5. Further, the relative order of subunits within the channel contributes to their functional properties.