Evidence of involvement of GIRK1/GIRK4 in long-term desensitization of cardiac muscarinic K⁺channels

Abstract

The cardiac M2 muscarinic receptor/G protein/K+ channel system was studied in neonatal rat atrial cells cultured with and without 10 microM carbachol (CCh) for 24 h. Channel activity in CCh-pretreated cells was substantially reduced as a result of long-term desensitization regardless of whether the channel was activated by ACh in cell-attached patches or GTP in inside-out patches. Channel activity in CCh-pretreated cells was also low when the receptor was bypassed and the G protein and channel were directly activated by [gamma-S]GTP or both the receptor and G protein were bypassed and the channel was directly activated by trypsin. Finally, in CCh-pretreated cells, the whole cell K+ current was low when the channel was activated via the independent adenosine receptor. This suggests that the channel is involved in long-term desensitization. However, in CCh-pretreated cells, although the receptor was internalized, there was no internalization of the channel. We suggest that the function of the muscarinic K+ channel declines in long-term desensitization of the cardiac M2 muscarinic receptor/G protein/K+ channel system.

Role of internalization of M2muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+current in heart

“…1 and 2). The first time constant compares well with previous measurements of the time constant of desensitization of I K,ACh , e.g., 2.5 min in guinea pig atrial cells (38) and 1.2 min in rabbit sinoatrial node cells (12), and the second time constant corresponds to a much slower process of desensitization of I K,ACh described by Bünemann et al (3) and Shui et al (30,32). Internalization of the M 2 receptor was equally as rapid: under similar conditions, M 2 -receptor labeling in the cell membrane declined by ϳ83% over a double-exponential time course with time constants of 2.9 Ϯ 1.7 and 51.8 Ϯ 26.3 min (Fig.…”

“…Although the results from the present study suggest that slow desensitization of I K,ACh is largely the result of the M 2 receptor, other possible factors cannot be excluded. Our group (32) has previously shown that, after a 24-h exposure to 10 M CCh, activity of the ACh-activated K ϩ channel in rat atrial cells is greatly reduced even if the M 2 receptor is bypassed and the channel is activated directly by GTP␥S (32). This suggests that, as well as the receptor, the G protein and/or the channel is altered during the desensitization process.…”

“…Approximately 9-wk-old (young adult) male rats (250 -300 g) and neonatal rats (1-3 days old) were killed humanely according to the United Kingdom Animals (Scientific Procedures) Act (1986); in addition, the investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1996). Atrial cells were isolated and prepared as described previously (27,32). The adult atrial cells were normally incubated in Tyrode solution (in mM: 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.5 CaCl2, 0.4 NaH 2PO4, 10 glucose, 20 taurine, 10 creatine, 5 HEPES, pH 7.3 with NaOH).…”

“…In addition, there is evidence that they may cause M 2 receptor uncoupling (21,22). In the heart, during exposure to agonist, the M 2 receptor is internalized (26,32). However, it is not clear whether an arrestin is involved.…”

Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

“…These findings suggest that Kir3 channel cell membrane expression is regulated by both G-proteins and endocytosis via activation of the GPCR. Trafficking of the Kir3 channel with the muscarinic receptor was unexpected; for example, carbachol stimulation of muscarinic receptors in atrial myocytes decreased levels of surface receptors but not Kir3.1/3.4 channels (Shui et al, 2001). A similar mechanism of dual regulation of N-type voltage-gated Ca 2ϩ channels and opioid receptor-like receptors 1 was recently reported; agonist stimulation triggered the vesicular internalization of the both the receptor and Ca 2ϩ channel in rat dorsal root ganglion cells, providing a key mechanism for regulating Ca 2ϩ channel activity (Altier et al, 2006).…”