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Cloned muscarinic receptor subtypes expressed in A9 L cells differ in their coupling to electrical responses
S V Jones1, J L Barker, N J Buckley
1Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
Abstract:
The electrophysiological responses to cholinergic stimulation of four cloned muscarinic receptor subtypes (m1-m4) were studied in A9 L cells transfected with the expression plasmids of each of the different subtypes, using the tight-seal whole-cell recording technique. Cells transfected with m1 and m3 muscarinic receptor subtypes were hyperpolarized by acetylcholine (ACh), whereas m2- and m4-transfected cells did not respond to ACh concentrations of up to 1 mM. Stimulation of both m1 and m3 muscarinic receptor subtypes evoked outward currents in cells voltage-clamped at -50 mV, associated with an increase in membrane conductance. These outward currents were blocked by atropine but not by tubocurarine. The ACh-induced currents of m1- and m3-transfected cells primarily involved potassium ions, although chloride ions also contributed to a minor extent. The potassium and chloride conductances were blocked by barium or cobalt and by buffering the intracellular calcium to low levels with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, showing a dependence of these conductances on calcium. Thus, m1- and m3-transfected cells respond to ACh in a manner that is qualitatively similar, evoking calcium-dependent potassium and chloride conductances, whereas m2- and m4-transfected cells are not coupled to electrically detectable responses in A9 L cells.
Insights
Acetylcholine activates muscarinic receptors m1 and m3, causing calcium-dependent potassium and chloride currents. Muscarinic receptors m2 and m4 did not show electrophysiological responses in A9 L cells.
Area of Science:
- Neuroscience
- Molecular Biology
- Cellular Electrophysiology
Background:
- Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors crucial for neurotransmission.
- Four subtypes (m1-m4) exist, with distinct signaling pathways and physiological roles.
- Understanding subtype-specific responses is key to elucidating cholinergic signaling.
Purpose of the Study:
- To investigate the electrophysiological effects of acetylcholine on cloned muscarinic receptor subtypes (m1-m4).
- To characterize the ion conductances and signaling pathways activated by m1 and m3 muscarinic receptors.
Main Methods:
- Utilized the tight-seal whole-cell recording technique in A9 L cells.
- Transfected cells with expression plasmids for individual muscarinic receptor subtypes (m1-m4).
- Stimulated cells with acetylcholine (ACh) and measured electrophysiological responses, including currents and membrane conductance.
Main Results:
- Acetylcholine induced hyperpolarization and outward currents in m1- and m3-transfected cells.
- These responses were associated with increased membrane conductance, blocked by atropine, and involved potassium and chloride ions.
- The observed conductances were calcium-dependent and blocked by barium or cobalt.
- m2- and m4-transfected cells showed no significant electrophysiological response to acetylcholine.
Conclusions:
- Muscarinic receptor subtypes m1 and m3 couple to calcium-dependent potassium and chloride conductances in A9 L cells.
- Muscarinic receptor subtypes m2 and m4 do not elicit detectable electrophysiological responses in this cellular system.
- This study highlights distinct signaling capabilities of muscarinic receptor subtypes.