Inhibition of Ca²⁺ Channels and Adrenal Catecholamine Release by G Protein Coupled Receptors

Abstract

Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the “fight-or-flight” response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca²⁺ entry through voltage-gated Ca²⁺ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers (Gβγ) bind to and inhibit Ca²⁺ channels. Here I review the mechanisms by which GPCRs inhibit Ca²⁺ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca²⁺ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.

The Fight-or-Flight Response and Stimulus-Secretion Coupling in Adrenal Chromaffin Cells

Adrenal chromaffin cells play important physiological roles in both homeostatic regulation and the coordinated response to stress or danger—i.e., the “fight-or-flight” response. For example, heart rate and contractility are increased, vascular tone and blood flow to skeletal muscle are increased, insulin release is suppressed, and glucose is mobilized from the liver. These and a myriad of other effects prime the body for a survival response to either fight or flee an enemy or dangerous situation. These diverse physiological effects are mediated by release of catecholamines (epinephrine and norepinephrine) and a number of other biologically active peptides and hormones from chromaffin cells. While beneficial in the short term, inappropriate responsiveness to stressors and/or chronic stress can be harmful. For example, elevated catecholamine levels are closely correlated with hypertension (Takiyyuddin et al. 1993) and contribute to the morbidity and mortality of chronic heart failure (Floras 2003).

Chromaffin cells, like neurons, release transmitters by Ca²⁺-regulated exocytosis. Unlike most neurons, the cells are ideally suited for patch-clamp electrophysiology to record ion channel currents and changes in membrane capacitance that track vesicle exocytosis and endocytosis with high time resolution (Lindau and Neher 1988; Gillis 2000). Another technique that directly detects catecholamine release with exquisite resolution is carbon fiber amperometry (Wightman et al. 1991). Catecholamines released from the cell are rapidly oxidized generating an electrical current that is directly proportional to the number of catecholamine molecules. With suitable stimulation protocols transient current spikes reflecting catecholamine release from individual vesicular fusion events can be detected and analyzed. These approaches combined with other techniques such as total internal reflection fluorescence (TIRF) microscopy and photorelease of “caged” Ca²⁺ can be applied to cells or adrenal slices from wild-type and transgenic mice to provide detailed insight into the cellular and molecular mechanisms that control neurosecretion.

In situ, chromaffin cells are innervated by splanchnic nerve terminals that release acetylcholine (ACh) and other transmitters including PACAP. Activation of nicotinic ACh receptors on the chromaffin cells causes membrane depolarization/action potential firing, opening of voltage-gated Ca²⁺ channels (Ca²⁺ channels), and Ca²⁺ influx that triggers fusion of the vesicles with the plasma membrane (Boarder et al. 1987). Consequently, Ca²⁺ channels play pivotal roles in stimulus-secretion coupling and are important targets for mechanisms that control transmitter release. Significant advances have also been made toward identifying the protein–protein interactions involved in exocytosis (for recent reviews see Rizo and Rosenmund 2008; Sorensen 2009; Sudhof and Rothman 2009). It is generally accepted that SNARE proteins constitute the core fusion machinery. Syntaxin and SNAP25 on the plasma membrane along with synaptobrevin on the vesicular membrane form a coiled-coil structure (the ternary SNARE complex) that positions the vesicle in close apposition to the plasma membrane. When the SNARE complex “zippers-up” it provides the force required to fuse the two adjacent lipid bilayers. Several members of the synaptotagmin family serve as Ca²⁺ sensors for exocytosis (Chapman 2008), and in particular synaptotagmins 1 and 7 are involved in chromaffin cells (Schonn et al. 2008). Although the precise mechanisms are still being defined, synaptotagmin-1 binds the plasma membrane and the SNARE complex in a Ca²⁺-dependent manner inducing conformational changes that trigger the final fusion event. The SNAREs are also involved in other aspects of the secretory vesicle cycle and recently it was proposed that docking of vesicles in mouse chromaffin cells is mediated by binding of synaptotagmin-1 to binary t-SNARE complexes (de Wit et al. 2009).

G protein Coupled Receptors (GPCRs) in Chromaffin Cells

GPCRs play central roles in orchestrating the dynamic modulation of transmitter release. Heterotrimeric G proteins act as molecular switches to transduce extracellular ligand binding to the GPCR into intracellular signaling cascades. GPCRs are often characterized according to the family of Gα subunit to which they couple: Gs-coupled receptors typically elevate cAMP; Gi-coupled receptors inhibit cAMP; Gq-coupled receptors activate phospholipase C; while G₁₂ receptor signaling is less well defined. Agonist binding to the GPCR catalyzes the exchange of GDP to GTP on the G protein α-subunit (Gα) and both Gα and the Gβγ heterodimer signal to downstream effectors. Of note for this review, these Gβγ effectors include voltage-gated Ca²⁺ channels and SNARE proteins (see below).

Chromaffin cells express a wide variety of GPCRs that sense and respond to changes in the local environment and perhaps the overall physiological “status” of the animal through hormones and other blood borne signals. A common theme at chromaffin cells and synapses is feedback modulation, whereby the released transmitters not only convey information to downstream targets but also act in an autocrine manner to modulate subsequent secretory activity. In general, GPCRs that couple to Gi-type G proteins inhibit catecholamine release, whereas Gq-coupled receptors and Gs-coupled receptors potentiate catecholamine release. Autoreceptors for ATP (P2Y receptors), catecholamines (α-adrenergic), and enkephalin (μ-opioid receptors) all couple to Gi-type G proteins and inhibit Ca²⁺ channels and transmitter release (Albillos et al. 1996b; Currie and Fox 1996; Harkins and Fox 2000; Powell et al. 2000; Ulate et al. 2000; Brede et al. 2003). Conversely, elevation of cAMP by Gs-coupled GPCRs (e.g., D1 dopamine, or β-adrenergic) can augment electrically evoked catecholamine release by increasing Ca²⁺ influx through L-type Ca²⁺ channels and/or direct protein kinase-A mediated phosphorylation of the exocytotic machinery (Artalejo et al. 1990; Carabelli et al. 2003; Nagy et al. 2004; Villanueva and Wightman 2007). Gq-coupled GPCRs (e.g., H1 histamine receptors) can release Ca²⁺ from intracellular stores and promote influx of extracellular Ca²⁺ to evoke catecholamine release. H1 receptors can also potentiate catecholamine release through generation of diacylglycerol which binds munc13 to increase the size of the readily releasable pool of vesicles (Bauer et al. 2007). PACAP/PAC1 receptors also play important roles in stimulating catecholamine release, particularly during periods of stress (Hamelink et al. 2002; Kuri et al. 2009).

Voltage-Gated Ca²⁺ Channels

As outlined above Ca²⁺ channels play pivotal roles in stimulus-secretion coupling and a multitude of other cellular functions. In large part this is due to their role in transducing the electrical inputs of excitable cells into biochemical outputs by influx of the ubiquitous second messenger Ca²⁺. The channels are hetero-oligomeric protein complexes containing a pore forming α1 subunit, a cytoplasmic β-subunit, a membrane associated α2δ-subunit, and perhaps a γ-subunit (Catterall 2000). Ten genes encode three major families of the pore forming α₁ subunit: four Ca_V1 (L-type channels), three Ca_V2 (P/Q-type, N-type, and R-type channels), and three Ca_V3 (T-type channels). The Ca_V3 family is referred to as low-voltage activated channels because they require only modest membrane depolarization to open. Ca_V1 and Ca_V2 channels are referred to as high-voltage activated as they typically require stronger membrane depolarization to open, although Ca_V1.3 and Ca_V2.3 channels activate at relatively hyperpolarized potentials. Indeed Ca_V1.3 channels play an important role in determining the spontaneous electrical activity of mouse chromaffin cells (Marcantoni et al. 2010). The pore forming α₁-subunit consists of four homologous repeats (domains I–IV) each with six transmembrane spanning α-helical segments (S1–S6) and a ‘P-loop’ between S5 and S6. The intracellular regions of the channel including the N- and C-termini and the cytoplasmic loops connecting the domains I–IV are important for interaction with other proteins including Gβγ and regulatory mechanisms such as phosphorylation. Alternative splicing of the α1 subunit can increase functional diversity, for example by introducing a site for tyrosine kinase phosphorylation in the C-terminus of Ca_V2.2 (Gray et al. 2007). Four genes encode β-subunits, with multiple splice variants for each, four genes encode α₂δ subunits, and ten putative γ subunit genes exist. Little is known about the role of γ subunits and it is not clear that they functionally associate with the channels in neurons and neuroendocrine cells. Both the α2δ and β subunits increase the expression and modulate activation and inactivation kinetics of Ca_V1 and Ca_V2 channels. The β-subunit might also be targeted by kinases to modulate channel activity (Grueter et al. 2006) and the α2δ subunits can bind extracellular ligands and drugs including thrombospondin and gabapentin/pregabalin (Davies et al. 2007; Eroglu et al. 2009).

Chromaffin cells can express channels from all three Ca_V families. Cell-to-cell and species-to-species variability in the relative expression of the Ca²⁺ channels has been reported by several groups (Garcia et al. 2006). Also of note, different channel types can be recruited by physiological or pathophysiological stressors. For example, Ca_V3 channels are typically not seen in recordings from adult chromaffin cells, but hypoxia or sustained β-adrenergic receptor signaling can increase plasma membrane expression of these channels and thereby shift the voltage-dependence of Ca²⁺ entry and exocytosis to more hyperpolarized potentials (Novara et al. 2004; Carabelli et al. 2007). Some studies have reported preferential coupling of specific channels to secretion or distinct vesicle pools, while others suggest the channels couple to secretion with similar efficiency (Artalejo et al. 1994; Engisch and Nowycky 1996; Lukyanetz and Neher 1999; Alvarez et al. 2008). The contribution of specific channel types can also change in an activity-dependent manner (Villarroya et al. 1999; Chan et al. 2005; Polo-Parada et al. 2006). Of note for this review, the Ca²⁺-channel subtypes are differentially modulated by G proteins and other second messenger pathways (Fox et al. 2008; Marcantoni et al. 2008).

Modulation of Ca²⁺ Channels by G Protein βγ Subunits

Inhibition of Ca²⁺ channels by neurotransmitter receptors is now recognized to be a widespread and important mechanism that controls neurotransmitter and hormone release (for reviews see Dolphin 2003; Tedford and Zamponi 2006; Catterall and Few 2008; Stephens 2009). Inhibition by GPCRs predominantly targets Ca_V2.1 (P/Q-type) and Ca_V2.2 (N-type) channels although Ca_V1 and Ca_V3 channels can also be inhibited, albeit by different molecular mechanisms. Several distinct signaling pathways converge on Ca_V2 channels but the most common and best understood mechanism is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the channel. Gβγ reduces the peak amplitude of the whole cell Ca²⁺-channel current (I_Ca), and produces characteristic shifts in channel gating: the voltage-dependence of activation is shifted to more depolarized potentials and activation kinetics are slowed; inhibition is diminished at depolarized membrane potentials; a conditioning prepulse to depolarized potentials can relieve most of the inhibition and normalize channel kinetics (hence the inhibition is termed voltage-dependent). In addition to being a defining biophysical signature, voltage-dependent relief of the inhibition (also termed facilitation) is physiologically relevant and can occur at least to some extent during high frequency trains of action potential-like waveforms (Womack and McCleskey 1995; Brody et al. 1997; Currie and Fox 2002). Inhibition is mediated by direct binding of Gβγ to the α₁ subunit of the channel, primarily the intracellular loop between domains I and II with other regions in domain I, the N-terminal, and C-terminal also implicated (Tedford and Zamponi 2006). These aspects have been incorporated into models in which the channels exhibit two functional gating states: “willing” in absence of bound Gβγ and “reluctant” when Gβγ is bound (Bean 1989; Elmslie et al. 1990; Colecraft et al. 2000; Lee and Elmslie 2000). Voltage-dependent relief of the inhibition by a prepulse or train of action potential-like stimuli is thought to reflect transient dissociation of Gβγ from the channel at depolarized potentials. At the single channel level Gβγ increases the latency to first opening of the channel in response to membrane depolarization with little impact on other parameters (Carabelli et al. 1996; Patil et al. 1996). Brief channel openings (from the “reluctant” state) have been reported to occur in N-type channels but the probability of such events is low (Colecraft et al. 2000; Lee and Elmslie 2000).

Both N-type and P/Q-type channels are subject to Gβγ mediated inhibition. However, the amplitude P/Q-type I_Ca is reduced to a lesser extent than N-type I_Ca (Currie and Fox 1997) and the inhibition of P/Q-type I_Ca can be relieved more easily by trains of action potential-like stimuli (Currie and Fox 2002). These differences are likely due to lower affinity binding of Gβγ at the P/Q-type channel. Protein kinase C can phosphorylate the I–II linker region of Ca_V2.2 (N-type) channels and thereby diminish binding of Gβ1-containing Gβγ heterodimers, but not heterodimers containing other Gβ subunits (Cooper et al. 2000). More recently it has been shown that Gβγ can reduce inactivation of N-type channels (McDavid and Currie 2006; Weiss et al. 2007). The molecular mechanisms that underlie voltage-dependent inactivation of Ca²⁺ channels are not fully understood but might involve a “hinged lid” type mechanism, with the intracellular loop connecting domains I and II of α₁-subunit serving as the “inactivation gate” (Stotz and Zamponi 2001). Intriguingly, the I–II loop is also a primary binding site of Gβγ on the channel (De Waard et al. 1997; Herlitze et al. 1997). Perhaps binding of Gβγ disrupts movement of this putative inactivation gate, or its interaction with other channel domains, but this remains to be determined.

Voltage-dependent inhibition by Gβγ demonstrates substantial plasticity and crosstalk with other signaling pathways that control I_Ca. For example, the extent of inhibition and the ability for high frequency bursts of action potentials to relieve the inhibition will depend upon the relative expression of N- and P/Q-type channels in a particular cell or synapse. Concomitant activation of PKC might selectively diminish inhibition of N-type channels, but only when the Gβγ heterodimer contains Gβ1-subunits. Due to reduced inactivation Gβγ alters the temporal profile as well as the absolute amount of Ca²⁺ influx during trains of action potential-like stimuli. Thus, Ca²⁺ channels integrate multiple convergent signals, both electrical and chemical (2nd messengers), to precisely tune Ca²⁺ entry for a given cellular context.

Voltage-dependent inhibition of N- and P/Q-type I_Ca is prominent in adrenal chromaffin cells and thought to underlie potent feedback inhibition of secretory activity. Several GPCRs have been shown to inhibit I_Ca including α-adrenergic, opioid and P2Y purinergic receptors (Diverse-Pierluissi et al. 1991; Kleppisch et al. 1992; Gandia et al. 1993; Twitchell and Rane 1993; Albillos et al. 1996a, b; Currie and Fox 1996). These receptors are of particular note because large dense core vesicles store and release catecholamines, ATP/ADP, and enkephalins, along with several other peptides and hormones. A variety of experiments have been performed to assess the impact of endogenous transmitters in chromaffin cells, and the consensus from these studies is that autocrine/paracrine inhibition of I_Ca is mediated by P2Y purinergic receptors and μ/δ opioid receptors (for review see Garcia et al. 2006). In contrast α2 adrenergic receptor modulation of I_Ca is not consistently seen in chromaffin cells. The inhibition produced by ATP is potent (EC₅₀ ~ 0.5 μM) (Currie and Fox 1996), shows little desensitization, and is likely mediated by a P2Y₁₂ receptor subtype (Ennion et al. 2004). Given the high concentration of ATP in the large dense core vesicles it is likely that autocrine purinergic modulation will potently and maximally inhibit I_Ca in the intact adrenal.

Voltage-independent inhibition of Ca_V2 channels also exists and likely represents a conglomerate of several distinct mechanisms. In chromaffin cells purinergic and opioid receptors can produce voltage-independent inhibition by a mechanism involving neuronal Ca²⁺ sensor-1 and src kinase (Weiss and Burgoyne 2001) and Gq-coupled H1 histamine receptors produce voltage-independent inhibition of Ca_V2 (Currie and Fox 2000). Direct interaction of GPCRs and N-type Ca²⁺ channels has been reported to lead to internalization of the channel when the activated GPCR is endocytosed (Altier et al. 2006) but it is not known if a similar mechanism exists in chromaffin cells.

G proteins activated by purinergic and opioid autoreceptors can also directly inhibit L-type I_Ca in chromaffin cells. This pathway is rapid, direct/membrane delimited, but voltage-independent (Hernandez-Guijo et al. 1999). Depending on the species, L-type channels contribute from ~10 to 40% of the whole cell I_Ca in chromaffin cells so play important roles in stimulus-secretion coupling (Garcia et al. 2006). Ca_V1.3 channels also play an important role in the basal firing properties of mouse chromaffin cells, so it will be interesting to see if these channels are inhibited by GPCRs and how this might impact electrical firing. Until recently it was not thought that Ca_V3 channels were modulated by GPCRs, but it has now been shown that Ca_V3.2 channels are inhibited by D1 dopamine and corticotropin-releasing factor type 1 receptors, the latter in adrenal cortical cells (Wolfe et al. 2003). At least some of these pathways involve direct effects of Gβγ, albeit in a voltage-independent manner. It will be interesting to determine if Ca_V3.2 channels upregulated in chromaffin cells by hypoxia or prolonged β-adrenergic stimulation are subject to modulation by endogenous GPCRs.

Inhibition of Catecholamine Release by Gβγ

The link between purinergic/opioid receptor-mediated inhibition of I_Ca and transmitter release in chromaffin cells has been shown by several groups (Harkins and Fox 2000; Powell et al. 2000; Ulate et al. 2000; Ennion et al. 2004). These studies used changes in membrane capacitance to assay exocytosis and concluded that inhibition of I_Ca is the dominant (if not the only) mechanism that inhibits exocytosis (but see Lim et al. 1997). More recently two studies in rat (Chen et al. 2005) or bovine chromaffin cells (Yoon et al. 2008) concluded that acute activation of P2Y or μ-opioid receptors, or direct application/transfection with Gβγ can inhibit catecholamine release independent from Ca²⁺ channel modulation. Catecholamine release was monitored using carbon fiber amperometry and evoked by methods that bypass Ca²⁺ channels (releasing Ca²⁺ from intracellular stores, applying Ca²⁺ directly through a patch pipette, or using the Ca²⁺ ionophore ionomycin). In both studies G proteins reduced the charge of individual amperometric spikes suggesting that, on average, the number of catecholamine molecules released by each vesicular fusion event was reduced. In bovine cells the number of amperometric spikes was also reduced.

The reduction of spike charge could reflect less catecholamine loaded into the vesicles, although the rapidly reversible effect of GPCR activation suggests this is less likely. Alternatively, Gβγ might preferentially inhibit release of a distinct subset of larger vesicles, and there is evidence for two populations of vesicles in mouse chromaffin cells based on estimates of vesicle size and the charge of amperometric spikes (Grabner et al. 2005). Another possibility discussed in both papers is that Gβγ might shift the mode of exocytosis and favor the occurrence of transient (kiss-and-run) rather than full fusion events. In classical, full fusion exocytosis the vesicle collapses into the plasma membrane thereby emptying the entire contents into the extracellular space. Transient events can also occur in which a fusion pore opens and then re-closes, thus maintaining the integrity of the vesicle and potentially resulting in partial release of the vesicle contents (for reviews see Harata et al. 2006; He and Wu 2007). Work from lamprey reticulospinal synapses supports the idea that GPCRs can modulate the mode of exocytosis (Photowala et al. 2006; Gerachshenko et al. 2009). In chromaffin cells, dynamic shifts from transient fusion to full collapse events have been reported to occur with increased stimulation frequency/Ca²⁺ entry (Elhamdani et al. 2001, 2006; Fulop et al. 2005; Fulop and Smith 2006). This has the potential to impact the identity as well as the amount of transmitter release from chromaffin cells because the narrow fusion pore in transient events might limit release of larger peptide transmitters by a simple size exclusion mechanism (Fulop et al. 2005). It is also interesting to note that concomitant activation of PKC prevented the effects of Gβγ on spike charge (“quantal size”) (Chen et al. 2005). Activation of PKC has also been implicated in the shift from transient to full fusion events during stimuli that mimic acute stress (15 Hz trains of action potential-like stimuli) (Fulop and Smith 2006). Therefore, it is possible that there are opposing actions of Gβγ and PKC on the exocytotic machinery to precisely control fusion pore kinetics.

The molecular targets that underlie these novel effects on catecholamine release remain unclear, but one plausible target is the core fusion machinery. Gβγ can bind to syntaxin-1A, synaptobrevin, SNAP25 and the ternary SNARE complex in vitro (Jarvis et al. 2002; Blackmer et al. 2005). Moreover, Gβγ and Ca²⁺-bound synaptotagmin-1 compete for binding to the SNARE complex in vitro (Yoon et al. 2007). In addition to being Ca²⁺ sensors for exocytosis, synaptotagmins have been implicated in fusion pore modulation (Wang et al. 2001; Bai et al. 2004; Moore et al. 2006), and vesicle docking in mouse chromaffin cells is mediated by interaction of synaptotagmin-1 with binary t-SNARE complexes (de Wit et al. 2009). Therefore, it is conceivable that Gβγ could modulate multiple facets of exocytosis through interactions with SNARE proteins. Of course Gβγ is known to interact with an increasing number of downstream effectors. Development of molecular tools to dissect the various targets of Gβγ will be important to tease apart the precise mechanisms and relative contribution of Ca²⁺ channels and other targets to the overall control of catecholamine release by Gβγ.

Concluding Remarks

It is clear that G proteins utilize multiple signaling pathways to control the timing and amount of Ca²⁺ entry in chromaffin cells. This and potentially other mechanisms that directly target the exocytotic machinery precisely regulate transmitter release. Clearly cellular context matters and must be considered as this field moves forward. As outlined above, Gβγ-mediated inhibition of I_Ca can be altered depending on the electrical firing of the cell, either through effects on inactivation of the channels or voltage-dependent relief of Gβγ binding. Concomitant activation of PKC by GPCRs and/or Ca²⁺ entry might antagonize inhibition of N-type I_Ca and the effects of Gβγ on the quantal size of release events. Another interesting consideration is cellular remodeling under physiological or pathophysiological conditions and how this might impact G protein control of transmitter release. This might involve changes in the Ca²⁺ channel subtype (as shown for hypoxia or sustained adrenergic receptor stimulation), or changes in the number or type of GPCRs expressed on the plasma membrane. Many other proteins can also regulate G protein signaling. Recent reports suggest that expression of G protein coupled receptor kinase 2 (GRK2) is increased in the adrenal gland in rodent models of heart failure (Lymperopoulos et al. 2007). GRK2 is involved in desensitization of α2-adrenergic and other GPCRs and it was suggested that increased GRK2 expression resulted in diminished feedback inhibition of catecholamine release. Reducing GRK2 expression in the adrenal gland decreased circulating catecholamines and improved heart function in these rodent models. Thus targeting feedback regulatory mechanisms in adrenal chromaffin cells might prove to be a useful therapeutic strategy in future.