Receptor-independent Activation of Atrial Muscarinic Potassium Channels in the Absence of Nucleotides

Abstract

The removal of nucleotides from the solution bathing the inner face of excised patches of frog atrial membranes was found to activate muscarinic K ؉ channels in the absence of agonists. Channel activation was also observed in Mg 2؉-free solutions and blocked by low (0.1-10 M) concentrations of GDP or GTP. After full activation was achieved, channel openings were abolished by the application of GDP-bound G␣ i2 but were not affected by exogenous G␤␥ dimers, suggesting that effector activation is a consequence of the liberation of ␤␥ subunits from endogenous G proteins. The process of channel activation in the absence of nucleotides seems to be receptor-independent, because it is not influenced by muscarinic receptor agonists and antagonists or by treatment with uncoupling agents such as pertussis toxin or N-ethyl maleimide. Taken together, these results suggest that the loss of GDP from the G protein nucleotide binding site promotes its uncoupling from receptors and destabilizes the G␣ (empty) ␤␥ heterotrimer. Therefore, the nucleotide-free form of G proteins has some of the characteristics of the GTP-bound, activated form.The muscarinic K ϩ channel (K ACh ) 1 is an inwardly rectifying, potassium-selective channel whose activation by agonistbound receptors is mediated by the ␤␥ subunits of a pertussis toxin (PTX)-sensitive GTP-binding protein (reviewed in Ref. 1). In the heart, the primary components of this system of signal transduction have been identified. These include the muscarinic m 2 receptor, heterotrimeric G proteins of the G i subfamily, and the heterotetrameric channel formed by K ir 3.1 (GIRK1) and K ir 3.4 (GIRK4 or CIR) subunits (2-5). Several neurotransmitter-regulated channels share the basic structural and functional features of atrial K ACh channels; therefore, the latter are considered a prototype for the study of G protein-channel interactions (6). In addition, because patch clamp methods allow the study of signal transduction with high time resolution and under circumstances in which the composition of the milieu can be strictly controlled, atrial muscarinic K ϩ channels can also be viewed as rapid, selective, and sensitive monitors of G protein function (7,8). In the present work, we used excised, inside-out patches of atrial membranes to examine how G proteins might be affected by the total absence of intracellular nucleotides. Our results provide new insights into the behavior of a nucleotide-free form of G proteins that appears regardless of receptor occupancy and suggest that the empty ␣ subunit shares some characteristics of the activated, GTP-bound form. EXPERIMENTAL PROCEDURESAtrial myocytes were obtained from bullfrog hearts by digestion with collagenase and trypsin (9). Pertussis toxin was a gift from Dr. Erik Hewlett (University of Virginia). G protein subunits ␣ i2 -GDP and ␤␥ were a gift from Dr. S. Graber (West Virginia University). Protease inhibitors and nucleotides were from Boehringer Manheim, except for Mg⅐ATP, which was from Sigma. All other chemicals were of the highest p...

Modulation of rat atrial G protein‐coupled K+ channel function by phospholipids

“…(6) After complete rundown of ATP‐sensitive K + channels, a process that has been attributed to depletion of PIP/PIP 2 in the membrane (Hilgemann & Ball, 1996; Fan & Makielski, 1997), we could easily activate K ACh channels with GTP (with ACh in the pipette) or GTPγS. In support of this, it was reported recently that K ACh channels could be activated via G protein even in the complete absence of ATP and Na + (Otero et al 1998). Together, these observations suggest strongly that ATP and PIP/PIP 2 use separate signalling pathways to modulate the G protein‐gated K + channel.…”

Spontaneous release of GDP from G i proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes

“…This channel activation was blocked upon addition of GDP and GDP-bound G αi2 (Otero et al 1998). The authors concluded that the nucleotide-free form of G proteins has some of the characteristics of the GTP-bound, activated form.…”

Tetraethylammonium potentiates the activity of muscarinic potassium channels in guinea‐pig atrial myocytes

“…2). Spontaneous run‐up of K ACh activity in nucleotide‐free solutions has also been reported for excised patches from frog atrial myocytes at 20°C (Otero et al 1998). In those experiments, the increase in activity was much smaller ( NP o < 0.05) than we observed in excised patches from guinea‐pig myocytes at 35°C and was attributed to spontaneous release of βγ subunits.…”