Structural elements in the Girk1 subunit that potentiate G protein–gated potassium channel activity

Abstract

G protein-gated inwardly rectifying K + (Girk/K IR 3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA B receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptorindependent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity.ion channel | electrophysiology | mutagenesis | baclofen

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K+(GIRK) channels by the novel anxiolytic drug, ML297

“…promote levels of GIRK2-containing channels on the cell surface comparable to wild-type GIRK1 (17). Here, we found that, whereas MPD activated GIRK-dependent responses in all transfected cells, ML297 sensitivity required the P-M2 domain of GIRK1, which contains the pore helix/K + selectivity filter and second membrane-spanning domain (Fig.…”

“…Importantly, whereas ML297 had negligible effects on holding current in neurons from Girk1 −/− mice, baclofen evoked reliable (albeit small) currents in these neurons ( Fig. 3 A and B), likely attributable to activation of residual GIRK1-lacking channels (17).…”

“…Although it cannot form functional homomers (12), GIRK1 is an integral subunit of the cardiac GIRK channel and most neuronal GIRK channels (13,14). GIRK1 confers robust basal and receptordependent activity to GIRK heteromers, attributable in part to unique residues in the pore and second transmembrane domain (15)(16)(17). The intracellular C-terminal domain also contributes to the potentiating influence of GIRK1 on channel activity, likely due to the presence of unique structures that modify the interaction between the channel and Gβγ, Gα, and PIP 2 (1)(2)(3).…”

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

“…1C, for an example), takes part in several aspects of GIRK regulation. First, it regulates both basal and neurotransmitter-evoked activity of the channel Kahanovitch et al, 2014;Wydeven, Young, Mirkovic, & Wickman, 2012). In particular, a single residue within the distal CT domain (Q404) selectively influences I evoked .…”

GIRK Channels

“…The composition of GIRK channels can also vary across cell types, even within a single brain region (Aguado et al, 2008;Cruz et al, 2004;Fernandez-Alacid et al, 2009). Given that GIRK channels composed of different subunits exhibit distinct functional properties ( Jelacic et al, 2000;Jelacic, Sims, & Clapham, 1999;Schoots et al, 1999;Wydeven, Young, Mirkovic, & Wickman, 2012), this diversity allows for varied GIRKmediated signaling across brain regions and cell types (for a detailed discussion of GIRK channel diversity, see Chapter 5).…”