Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells

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.

Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels

“…Acetylcholine stimulated T-type currents in NIH 3T3 cells that had been transfected with recombinant m3 and m5 muscarinic receptors (322). Stimulation was modest (25%) and was accompanied by a leftward shift in the I-V relation.…”

Selective Inhibition of Cav3.3 T-type Calcium Channels by Gαq/11-coupled Muscarinic Acetylcholine Receptors

“…Our results using exogenous expression of cloned T-type Ca 2ϩ channels indicates that M1 receptor activation has a robust inhibitory effect on Cav3.3 currents and has either no effect or a small stimulation on both Cav3.1 and Cav3.2 currents. Similarly, experiments examining native Cav3.2 Ca 2ϩ channels in NIH3T3 cells transiently transfected with mAChRs demonstrated that M1 receptor activation had either no effect or a stimulatory effect if a PKC inhibitor was applied (27). Active G␤ 2 ␥ subunits have been shown to specifically inhibit Cav3.2 currents (42,55), and the lack of inhibition of Cav3.2 channels by M1 receptors in our study is likely because of the absence of any functional coupling between M1 receptors and G␤ 2 proteins (56).…”

“…2ϩ Channel Isoforms-Examination of the literature shows that activation of mAChRs can result in multiple effects on native T-type Ca 2ϩ currents, including causing stimulation (26,27,53), inhibition (28), or having no effect (54). Given the heterogeneous nature of native low threshold Ca 2ϩ currents, without investigating interactions between specific mAChR gene products and specific T-type Ca 2ϩ channel isoforms, the published differences in modulation FIGURE 6.…”

“…Neurotransmitters such as acetylcholine have been shown to either attenuate or stimulate low threshold Ca 2ϩ currents depending on the type of native cells examined, and sometimes multiple forms of modulation can be observed within the same cell type (25)(26)(27)(28)(29). Multiple T-type Ca 2ϩ channel subtypes are expressed in most native cells (30,31), although pharmacological tools with the specificity needed to separate these currents have not been generated.…”

Voltage-operated calcium channels in male germ cells

“…There have been relatively few reports of regulation of LVA channel currents, although it is clear that such regulation can occur, both in excitable and non-excitable cells (Barrett et al, 1991;Chesnoy-Marchais and Fritsch, 1994;Pemberton et al, 2000). The T-like current of mouse male germ cells is subject to control by tyrosine phosphorylation-dephosphorylation.…”