Search

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein [beta][gamma], subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein [beta][gamma] subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K+ ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., lannaccone, S. T., Brunt, E., Barohn, R., etal. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.

Slow nonselective cation conductances play a central role in determining the excitability of many neurons, but heretofore this channel type has not been analyzed at the single-channel level. Neurotensin (NT) excites cultured dopaminergic neurons from the ventral tegmental area primarily by increasing such a cation conductance. Using the outside-out configuration of the patch clamp, we elicited single-channel activity of this NT-induced cation channel. Channel activity was blocked by the nonpeptide NT antagonist SR48692, indicating that the response was mediated by NT receptors. The channel opened in both solitary form and in bursts. The reversal potential was -4.2 [+ or -] 1.7 mV, and the elementary conductance was 31 pS at -67 mV with [[[Na.sup.+]].sub.o] = 140 mM, [[[Cs.sup.+]].sub.o] = 5 mM, [[[Na.sup.+]].sub.i] = 88 mM, and [[[Cs.sup.+]].sub.i] = 74 mM. Thus, the channel was permeable to both [Na.sup.+] and [Cs.sup.+]. From these characteristics, it is likely that this channel is responsible for the whole-cell current we studied previously. In guanosine 5[prime]-[[Gamma]-thio]triphosphate-loaded cells, NT irreversibly activated about half of the channel activity, suggesting that at least part of the response was mediated by a G protein. Similar channel activity could be induced occasionally in the cell-attached configuration by applying NT outside the patch region.

Abstract: HEK293 cells were transfected with cDNAs for Gβ1(W332A) [a mutant Gβ1], Gγ2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gβ1γ2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gβ1(W332A) suppressed this current. Most likely, Gβ1(W332A) inhibited the action of the endogenous Gβ. Such a dominant negative effect of Gβ1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gβ1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gβ from activating the channel. [Copyright &y& Elsevier]

Examines the role of glutamate residue of G protein-coupled inward rectifiers (GIRK) in the regulation of potassium channels. Localization of GIRK2 in the domain of C terminus; Mechanisms of GIRK and inward rectifiers trafficking to cell membranes; Interaction between GIRK2 and G protein ΒΓ.