Muscarinic K+ Channel in the Heart

Abstract

The membrane-delimited activation of muscarinic K+ channels by G protein βγ subunits plays a prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying K+ channels. Here, we demonstrate that muscarinic K+ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel, which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of Gβγ subunits to the channel was tested, using recombinant Gβ1γ5. Gβ1γ5 was able to control the equilibrium between the four gating modes of the channel in a manner consistent with binding of Gβγ to four equivalent and independent sites in the protein complex. Surprisingly, however, Gβ1γ5 lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K+ channel gating by Gβγ provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.

Coordination of Membrane Excitability through a GIRK1 Signaling Complex in the Atria

“…Membrane-associated proteins were solubilized according to a protocol previously used by us for purification of heterotrimeric G proteins (22). The membranes were incubated for 3 h in MEB buffer (20 mM HEPES, pH 8.0, 2 mM MgCl 2 , 1 mM EDTA, 100 mM NaCl, 10 mM ␤-mercaptoethanol, 1% sodium cholate, and 0.45% IGEPAL CA-630) with PI.…”

“…Single-channel Recordings and G Protein Purification-Unitary currents through K ACh channels were recorded from inside-out patches of neonatal rat atrial myocytes using a standard high resolution patchclamp method (23) as previously described (22). The composition of the bath solution was (in mM): 150 KCl, 5 EGTA, 5 glucose, 1.6 MgCl 2 , 5 HEPES (pH 7.4); the composition of the pipette solution was (in mM): 150 KCl, 1 CaCl 2 , 1.6 MgCl 2 , 5 HEPES (pH 7.4).…”

“…The modal behavior of G␤ 1 ␥ 7 -activated K ACh channels was analyzed in the context of a model that assumes four functional modes of a single K ACh channel arising from different occupancy of four equivalent and independent G␤␥ sensors in the channel, as previously described (22). The continuous records were divided into 400-ms consecutive segments, and the frequency of apparent openings, f, was calculated for each data segment.…”

“…G protein G␤ 1 ␥ 7 dimers were expressed and purified from Sf9 cells in the Laboratory of Dr. Janet Robishaw as described previously (22).…”

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

“…G Protein-regulated Conformational States of the GIRK Channel-Measurements of single-channel or population currents in native and chimeric GIRK channels demonstrated that heterotetrameric GIRK channels assume a number of closed and open conformations with a variable number of G␤␥ molecules bound, from a closed, non-conducting state (interpreted as G␤␥-devoid), via intermediate states with one, two, or three G␤␥ molecules bound and correspondingly increasing open probability (P o ), to a fully G␤␥-activated state with four G␤␥ bound and the highest P o (27)(28)(29)(30). Analysis of i-FRET, when combined with biochemical and functional assays, extends our understanding of the rules and modes of G protein-GIRK interactions and of the regulation of channel conformation.…”