Presynaptic CaMKIIα modulates dopamine D3 receptor activation in striatonigral terminals of the rat brain in a Ca²⁺ dependent manner

Highlights

• Coactivation of dopamine D3 receptors with D1 receptors potentiates cAMP formation and GABA release in SNr.

• The potentiating effects of activating dopamine D3 receptors are antagonized by procedures that increase cytoplasmic Ca²⁺.

• The effects of increasing cytoplasmic Ca²⁺ are reversed by CAMKII antagonist KN-62.

• Dopamine D3 receptor activity is inhibited by binding to CAMKII.

Abstract

CaMKIIα is expressed at high density in the nucleus accumbens where it binds to postsynaptic D3 receptors inhibiting their effects. In striatonigral projections, activation of presynaptic D3 receptors potentiates D1 receptor-induced stimulation of cAMP production and GABA release. In this study we examined whether the presynaptic effects of D3 receptor stimulation in the substantia nigra reticulata (SNr) are modulated by Ca²⁺ activation of CaMKIIα. In SNr synaptosomes two procedures that increase cytoplasmic Ca²⁺, ionomycin and K⁺-depolarization, blocked the additional stimulation of cAMP accumulation produced by coactivating D3 and D1 dopamine receptors. The selective CaMKIIα inhibitor KN-62 reversed the blockade produced by ionomycin and K⁺-depolarization. Incubation in either Ca² -free solutions or with the selective Ca²⁺ blocker nifedipine, also reversed the blocking effects of K⁺-depolarization. Immunoblot studies showed that K⁺-depolarization increased CaMKIIα phosphorylation in a KN-62 sensitive manner and promoted CaMKIIα binding to D3 receptors. In K⁺-depolarized tissues, D3 receptors potentiated D1 receptor-induced stimulation of [³H]GABA release only when CaMKIIα was blocked with KN-62. In the presence of this inhibitor, the selective D3 agonist PD 128,907 reduced the ED₅₀ for the D1 agonist SKF 38393 from 56 to 4 nM. KN-62 also enhanced the effects of dopamine on depolarization induced [³H]GABA release. KN-62 changed ED₅₀ for dopamine from 584 to 56 nM. KN-62 did not affect D1 and D4 receptor responses. These experiments show that in striatonigral projections, CaMKIIα inhibits the action of D3 receptors in a Ca²⁺ dependent manner blocking their modulatory effects on GABA release. These findings suggest a mechanism through which the frequency of action potential discharge in presynaptic terminals regulates dopamine effects.

Introduction

Dopamine modifies neuronal function by interacting with two classes of G protein-coupled receptor families, the D1 family (that includes D1 and D5 receptors) typically coupled to Gs proteins and the D2 family (D2, D3, and D4 receptors) typically couple to Gi proteins (Neve et al., 2004). The rat D3 receptor (D3R) messenger translates a 446 amino acid protein that has 52% homology with the rat D2 receptor; 75% of the homology is in the transmembrane-spanning domains. D3Rs are preferentially expressed in the mesolimbic dopaminergic projection areas, including the nucleus accumbens (NAc) (Sokoloff et al., 1990; Bouthenet et al., 1991). In the nucleus accumbens D3Rs behave as typical D2 family receptors, i.e., they are coupled with Gi proteins inhibiting adenylyl cyclase (Schwartz et al., 1998). Recently it has been shown that D3R function in the NAc is markedly regulated by Ca²⁺/calmodulin-dependent protein kinase II α (CaMKIIα). In this structure CaMKIIα binds to D3Rs in a Ca²⁺-sensitive manner, phosphorylating the receptor and inhibiting its function (Liu et al., 2009).

D3Rs are also active in striatonigral nerve endings where they modulate GABA release (Cruz-Trujillo et al., 2013). In these structures the D3R signaling pathway is “atypical”. Instead of acting through coupling to Gi proteins; D3Rs enhance D1R-stimulated cAMP production and GABA release (Cruz-Trujillo et al., 2013). Since CaMKIIα is an abundant enzyme in the brain, especially at synaptic sites (Kelly et al., 1984), we examined whether it also modulates D3Rs signaling localized in presynaptic endings where it is mediated by an “atypical” pathway.

CaMKIIα activity depends on the level of cytoplasmic Ca²⁺ (Lisman et al., 2002). As cytoplasmic Ca²⁺ increases, the enzyme is removed from its autoinhibitory state and becomes activated by the binding of Ca²⁺ and calmodulin (CaM). Activated CaMKIIα phosphorylates its substrates as well as an autophosphorylation site within its autoregulatory domain. The autophosphorylation results in a Ca²⁺/CaM-independent kinase activity even after the initial Ca²⁺ stimulus subsides (Hudmon and Schulman, 2002; Colbran and Brown, 2004; Griffith, 2004).

In this study we evaluated whether procedures that markedly enhance cytoplasmic Ca²⁺ modify the effects of activating D3Rs in the striatal projections to the SNr via a CaMKIIα-mediated process. We concentrated, mostly, on the effects of K⁺-induced depolarization because this procedure triggers Ca²⁺-dependent transmitter release that is akin to the naturally occurring process (Bernath, 1992). Furthermore, K⁺ depolarization of neuronal cultures leads to CaMKIIα accumulation near active zones (AZs) (Tao-Cheng et al., 2006).

Regulation of dopaminergic receptors at presynaptic endings by CaMKIIα is very likely of functional relevance since neural activity modifies presynaptic CaMKIIα activity (see Shakiryanova et al., 2011). An account of our results has already been published in preliminary form (Arturo et al., 2010).