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Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and
F Vogalis1, M Ward, B Horowitz
1Department of Physiology, University of Nevada, School of Medicine, Reno 89557-0046, USA.
Abstract:
Functional coupling between muscarinic (m3) receptors and two voltage-gated K+ (Kv) channels (Kv1.2 and Kv1.5) cloned originally from canine colonic smooth muscle was studied using the Xenopus oocytes expression system and a mammalian cell line (COS cells). Oocytes were coinjected with cRNAs encoding the human m3 receptor and the Kv channel clones. COS cells were stably transfected with the hm3 cDNA and the cDNA encoding Kv1.5 channels. In oocytes coexpressing hm3 receptors and Kv channels, acetylcholine (ACh, 100 microM) decreased the whole-oocyte Kv channel current (IKv) by 72% over 20 min. ACh was equally effective at suppressing IKv1.2 as IKv1.5. In oocytes expressing only Kv channels phorbol esters (phorboldibutyrate) and phorbol dideconoate (10-30 nM) mimicked the action of ACh on IKv in oocytes coexpressing hm3 receptors. At the single-channel level, both ACh and phorbol dibutyrate applied to the extra-patch membrane reduced the open probability of Kv channels in the cell-attached patches without affecting single-channel conductance. In cotransfected COS cells, over a similar time course as in oocytes ACh suppressed whole-cell IKv1.5, but only by 30% and the effect was not reversible. These data indicate that stimulation of m3 receptors in cells that express Kv1.2 and Kv1.5 channels causes a poorly reversible decrease in the open probability of these channels.
Insights
Stimulating muscarinic 3 (m3) receptors reduces the activity of voltage-gated potassium (Kv) channels Kv1.2 and Kv1.5. This interaction affects channel open probability, impacting cellular function.
Area of Science:
- Molecular biology
- Cellular physiology
- Pharmacology
Background:
- Muscarinic (m3) receptors are G protein-coupled receptors involved in various physiological processes.
- Voltage-gated potassium (Kv) channels play critical roles in regulating cell membrane potential and excitability.
- Understanding the functional coupling between these two protein types is crucial for elucidating cellular signaling pathways.
Purpose of the Study:
- To investigate the functional coupling between human muscarinic 3 (m3) receptors and canine colonic smooth muscle Kv1.2 and Kv1.5 channels.
- To determine the effect of acetylcholine (ACh) stimulation on Kv channel activity when coexpressed with m3 receptors.
- To explore the underlying mechanisms of this functional interaction.
Main Methods:
- Utilized the Xenopus oocytes expression system for co-expressing m3 receptors and Kv channel cRNAs.
- Employed a mammalian cell line (COS cells) for stable transfection with m3 receptor and Kv1.5 channel cDNAs.
- Performed whole-cell and single-channel electrophysiology to measure Kv channel currents and open probability.
Main Results:
- Acetylcholine (ACh) significantly decreased whole-oocyte Kv channel current (IKv) by 72% in cells coexpressing m3 receptors and Kv1.2/Kv1.5 channels.
- Phorbol esters mimicked ACh's effect on IKv in oocytes expressing only Kv channels, suggesting a protein kinase C (PKC) pathway involvement.
- ACh and phorbol dibutyrate reduced Kv channel open probability at the single-channel level without altering conductance.
- In COS cells, ACh caused a less pronounced and poorly reversible suppression of IKv1.5.
Conclusions:
- Functional coupling exists between m3 receptors and Kv1.2/Kv1.5 channels.
- m3 receptor stimulation leads to a decrease in Kv channel open probability, mediated possibly through a PKC-dependent pathway.
- This interaction results in a poorly reversible reduction in Kv channel activity, impacting cellular excitability.