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Differential time course for desensitization to muscarinic effects on K+ and Ca2+ channels
K Mubagwa1, J C Gilbert, A J Pappano
1Department of Pharmacology, University of Connecticut Health Center, Farmington 06030.
Abstract:
The time course of muscarinic effects on K and Ca currents was investigated at 22-24 degrees C in guinea-pig atrial myocytes, using the whole-cell voltage clamp. At a holding potential of -40 or -50 mV, short exposures to 100 microM acetylcholine (ACh) or carbachol (CCh) reproducibly induced outward K currents (IK,ACh). During long exposures to these agonists, IK,ACh faded with time. In cells not dialysed with guanosine triphosphate (GTP), IK,ACh could dissipate completely following 15-20 min of agonist exposure. After agonist washout, lost sensitivity was not recovered. In cells dialysed with GTP (0.2-1 mM), IK,ACh still faded but normal sensitivity to agonists was restored with washout. Fade of IK,ACh was not prevented by intracellular heparin or dextran, excluding the involvement of either beta-adrenergic or muscarinic receptor kinase. IK,ACh induced by bethanechol or adenosine also faded, and subsequent CCh application after washout revealed a diminished response. Intracellular guanosine-5'-o-(3-thiotriphosphate (GTP gamma S) induced IK,ACh which faded, and subsequent exposure to CCh was without effect. Equally, after full desensitization with CCh, GTP gamma S failed to induce IK,ACh. The Ca current (ICa) was activated by voltage steps to 0 mV and increased with 1-3 microM isoproterenol. This increase could be reversed by CCh, even when IK,ACh had completely faded. Prolonged muscarinic agonist exposure sometimes also caused fade of the effect on ICa, which always occurred after loss of IK,ACh. The results show that desensitization is heterologous and may involve the guanine nucleotide-binding (G) protein. The differential desensitization to the effects on IK,ACh and ICa suggests the involvement of two different signalling pathways in the muscarinic control of K and Ca channels.
Insights
Muscarinic agonists like acetylcholine desensitize K+ currents (IK,ACh) in heart cells. This desensitization is heterologous, suggesting involvement of G proteins and distinct pathways for K+ and Ca2+ channel control.
Area of Science:
- Cardiovascular Physiology
- Cellular Electrophysiology
- Pharmacology
Background:
- Muscarinic receptors modulate ion channel activity in cardiac myocytes.
- Understanding the desensitization mechanisms of these currents is crucial for cardiac function.
- Previous studies have implicated various signaling pathways in receptor desensitization.
Purpose of the Study:
- To investigate the time course and characteristics of muscarinic effects on potassium (K) and calcium (Ca) currents in guinea-pig atrial myocytes.
- To elucidate the underlying mechanisms of desensitization for these ion currents.
- To determine if desensitization is specific to certain pathways or heterologous.
Main Methods:
- Whole-cell voltage clamp technique was employed on guinea-pig atrial myocytes.
- Cells were exposed to various muscarinic agonists (acetylcholine, carbachol, bethanechol) and other modulators (isoproterenol, GTP, GTPγS).
- Time course of outward K+ currents (IK,ACh) and Ca2+ currents (ICa) were measured, along with recovery after agonist washout.
Main Results:
- Short exposure to acetylcholine (ACh) or carbachol (CCh) induced outward K+ currents (IK,ACh), which faded with prolonged exposure.
- IK,ACh desensitization was reversible with GTP, but not without it, suggesting G protein involvement.
- Desensitization was heterologous, affecting both IK,ACh and ICa, with ICa desensitization occurring after IK,ACh loss.
- GTPγS induced IK,ACh that faded, and subsequent CCh had no effect, indicating desensitization upstream of G protein activation.
Conclusions:
- Muscarinic receptor desensitization in atrial myocytes is heterologous and likely involves guanine nucleotide-binding (G) proteins.
- Differential desensitization of IK,ACh and ICa suggests the involvement of at least two distinct signaling pathways in muscarinic control.
- These findings provide insights into the complex regulation of cardiac ion channels by muscarinic receptors.