Acetylcholine (ACh) released from the stimulated vagus nerve decreases heart rate via modulation of several types of ion channels expressed in cardiac pacemaker cells. Although the muscarinic-gated potassium channel I(KACh) has been implicated in vagally mediated heart rate regulation, questions concerning the extent of its contribution have remained unanswered. To assess the role of I(KACh) in heart rate regulation in vivo, we generated a mouse line deficient in I(KACh) by targeted disruption of the gene coding for GIRK4, one of the channel subunits. We analyzed heart rate and heart rate variability at rest and after pharmacological manipulation in unrestrained conscious mice using electrocardiogram (ECG) telemetry. We found that I(KACh) mediated approximately half of the negative chronotropic effects of vagal stimulation and adenosine on heart rate. In addition, this study indicates that I(KACh) is necessary for the fast fluctuations in heart rate responsible for beat-to-beat control of heart activity, both at rest and after vagal stimulation. Interestingly, noncholinergic systems also appear to modulate heart activity through I(KACh). Thus, I(KACh) is critical for effective heart rate regulation in mice.
GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (I KACh ) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein G ␥ subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique (ϳ125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flagtagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35 S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of ⌬374 -501 resulted in an intracellular GIRK1 subunit that produced normal I KACh -like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of I KACh in vivo.
Background Alternans of intracellular Ca2+ (Cai) underlies T-wave alternans, a predictor of cardiac arrhythmias. A related phenomenon, T-Wave Lability (TWL), precedes Torsade de Pointes (TdP) in patients and animal models with impaired repolarization. However, the role of Cai in TWL remains unexplored. Methods Action potentials (APs) and Cai transients, (CaTs) were mapped optically from paced Langendorff female rabbit hearts (n=8) at 1.2s cycle length, after AV node ablation. Hearts were perfused with normal Tyrode's solution then with dofetilide (0.5 μM) and reduced [K+] (2 mM) and [Mg2+] (0.5 mM) to elicit long QT type 2 (LQT2). Lability of EKG, voltage and Cai signals were evaluated during regular paced rhythm, before and after dofetilide perfusion. Results In LQT2, lability of Cai, voltage and EKG signals increased during paced rhythm, before the appearance of early afterdepolarizations (EADs). LQT2 resulted in AP prolongation and multiple (1-3) additional Cai upstrokes, while APs remained monophasic. When EADs appeared, Cai rose before voltage upstrokes at the origins of propagating EADs. Interventions (i.e. ryanodine and thapsigargin, n=3 or low [Ca]o and nifedipine, n=4) that suppressed Cai oscillations also abolished EADs. Conclusions In LQT2, Cai oscillations (CaiO) precede EADs by minutes, indicating that they result from spontaneous sarcoplasmic reticulum Ca2+ release rather than spontaneous ICaL reactivation. CaiO likely produce oscillations of Na/Ca exchange current, INCX. Depolarizing INCX during the AP plateau contributes to the generation of EADs by re-activating Ca2+-channels that have recovered from inactivation. TWL reflects CaTs and APs lability that occur before EADs and TdP.
Predictors of complications after lead extraction procedures include a higher number of extracted leads and the presence of defibrillator as opposed to pacemaker leads. A new paradigm of the removal of all leads not connected to a device may, therefore, reduce the risk of complications from lead extraction procedures and deserves to be tested prospectively.
The cardiac G protein-gated K ؉ channel, I KACh , is directly activated by G protein ␥ subunits (G ␥ ). I KACh is composed of two inward rectifier K ؉ channel subunits, GIRK1 and GIRK4. G ␥ binds to both GIRK1 and GIRK4 subunits of the heteromultimeric I KACh . Here we delineate the G ␥ binding regions of I KACh by studying direct G ␥ interaction with native purified I KACh , competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in G ␥ binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209 -225 or 226 -245, effectively competed for G ␥ binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting G ␥ binding and G ␥ activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226 -245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of G ␥ binding to I KACh and G ␥ activation of GIRK1/mutant GIRK4 channels. We conclude from this data that G ␥ binding to GIRK4 is critical for I KACh activation. Acetylcholine (ACh)1 secreted from the vagus nerve binds cardiac muscarinic receptors, initiating a sequence of events leading to slowing of heart rate (1-3). I KACh , an inwardly rectifying, K ϩ -selective channel, mediates part of this process by hyperpolarizing pacemaker cells in sinoatrial and atrioventricular nodes of the heart (for review, see Ref. 4). Muscarinic, adenosine, and other receptors (5) all catalyze the release of G ␥ subunits from pertussis toxin-sensitive heterotrimeric G proteins, which in turn directly activates the channel (6 -9). Cardiac I KACh is composed of two homologous inward rectifier K ϩ channel subunits, GIRK1 (9, 10) and GIRK4 (11), which form a heterotetramer consisting of two GIRK1 and two GIRK4 subunits (12). Similar complexes comprised of GIRK1 and GIRK2 (13-17), or GIRK1 and GIRK3 (13) appear to form neuronal G protein-gated K ϩ channels. Both channel subunits bind G ␥ with similar affinity (18 -22).An important finding that is crucial to understanding G ␥ regulation of I KACh is that G ␥ activates homomultimeric GIRK4 (11, 15) or homomultimeric GIRK2 (16) channels. This implies that the GIRK4 subunit alone contains the necessary elements required for activation. In contrast to GIRK2 and GIRK4, GIRK1 subunits are not functionally expressed as homomultimers (23-25), and this fact limits conclusions that can be drawn about GIRK1 contributions to G ␥ regulation.Initially, GIRK1 was thought to comprise the entire I KACh channel (9, 10), and initial studies on the mechanism of G ␥ activation of I KACh focused on GIRK1. Chimeric channels were constructed from GIRK1 and non-G protein-sensitive K ϩ channel inward rectifier (Kir) subunits to examine this problem. Results from the expression of these chimeras in Xenopus oocytes were used to conc...
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