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Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells
K E Pemberton1, L J Hill-Eubanks, S V Jones
1Department of Psychiatry, University of California, San Diego, La Jolla 92093-0603, USA.
Abstract:
The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 muscarinic receptor, the increased T-type channel activity appeared to be mediated via increased cAMP levels and subsequent activation of PKA. The lack of effect of the ml muscarinic receptor on the T-type calcium channel was probably due to the opposing actions of concomitant activation of both PKC and PKA. The physiological significance of these findings is discussed.
Insights
Muscarinic receptors m3 and m5 enhance T-type calcium channel activity, primarily through increased cyclic AMP (cAMP) and protein kinase A (PKA) pathways. The m1 receptor
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Physiology
Background:
- Muscarinic receptors (mAChRs) are G protein-coupled receptors involved in various physiological processes.
- T-type calcium channels play crucial roles in neuronal excitability and neurotransmitter release.
- The interaction between mAChRs and T-type calcium channels is not fully elucidated.
Purpose of the Study:
- To investigate the functional effects of stably expressed muscarinic receptor subtypes (m1-m5) on T-type calcium currents in NIH 3T3 cells.
- To elucidate the signaling pathways involved in mAChR-mediated modulation of T-type calcium channels.
Main Methods:
- Whole-cell patch-clamp electrophysiology to record T-type calcium currents.
- Stable transfection of NIH 3T3 cells with individual muscarinic receptor subtypes.
- Application of receptor agonists (acetylcholine, carbachol), subtype-specific antagonists (atropine), and signaling pathway modulators (forskolin, 8-bromo-cAMP, RpcAMPS, calphostin C, PDBu).
- Intracellular cAMP level measurements using immunoassay.
Main Results:
- Muscarinic receptors m3 and m5 stimulation increased T-type calcium current amplitude in a pertussis-toxin-insensitive manner.
- The m3 receptor-mediated increase involved enhanced cyclic AMP (cAMP) levels and protein kinase A (PKA) activation, shifting channel activation to more hyperpolarized potentials.
- Muscarinic receptors m2 and m4 stimulation decreased cAMP levels, while m1 receptor stimulation's effect on T-type currents was masked by opposing protein kinase C (PKC) and PKA pathways, but could be unmasked by PKC inhibition.
Conclusions:
- Muscarinic receptors m3 and m5 positively modulate T-type calcium channel activity, primarily via the cAMP/PKA pathway.
- Muscarinic receptor m1 can also modulate T-type calcium channels, but its effect is complex due to simultaneous activation of PKC and PKA.
- These findings reveal subtype-specific mechanisms of muscarinic receptor regulation of T-type calcium channels, with implications for neuronal function.