Ion Channel Modulation by G-Protein Coupled Receptors

The G-protein coupled receptors (GPCRs) are the largest and most versatile protein family in the mammalian genome. They interact with G-proteins to activate many intracellular signaling pathways and modulate ion channel activity. GPCRs modulate ion channels by two distinct pathways. The main focus of this review is the direct modulation of N-type, P/Q-type Ca²⁺ and K_ir3 K⁺ channels by the G_βγ subunit, the membrane delimited pathway. Since both ion channels and GPCRs play an important role in intracellular signaling, they have become targets in the search for therapeutic drugs for cancer, heart diseases, obesity and others.

The GPCRs Superfamily

Intracellular signaling, the cellular tool for communication, is a very complex and diverse process. This process relies on elaborate systems of proteins which enable cells to respond to a variety of stimuli in their internal or external environment.^1,2 Among these membrane proteins is the GPCRs Superfamily. They form the largest and most diverse family of cell surface receptors and proteins. For example, there are about 1000 GPCRs in neurons alone. The diversity of this superfamily is a result of the large number of members comprising this family, their ability to form different dimer combinations and their ability to respond to a multitude of stimuli, as well as by the large number of intracellular signaling pathways they activate.^2,3

Despite their structural and functional diversity, all GPCRs share a similar molecular architecture. They consist of seven transmembrane domains, linked by alternating intracellular and extracellular loops, an extracellular N-terminus and an intracellular C-terminus.³

An enormous amount of signal molecules, hormones, neurotransmitters, chemokines and local mediators, can activate different GPCRs with high specificity. GPCRs are associated with distinct classes of heterotrimeric GTP-binding proteins (G-proteins).^2-5

The G-proteins consists of three subunits: α, β and γ. To date, about 17 genes encoding the α-subunits, 5 for β-subunits and 12 for the γ- subunits, have been identified.^5,6 The interaction of G-proteins with GPCRs leads to the dissociation of the G_α subunit from the G_βγ, and either subunit may act as a downstream effector. The G_βγ acts as a dimer subunit, enhancing the receptor mediated signal by activating a distinct and diverse array of end targets such as enzymes and ion channels.^5-7

These signaling events are terminated following the reassociation of the G-protein heterotrimer. This is due to the intrinsic GTPase activity of the Gα subunit, which hydrolyzes bound GTP to GDP. This activity was shown to be accelerated by a group of proteins, GTPase-activating proteins that were named regulators of G-protein-Signaling (RGS). The RGS exert their action by interacting directly with the G_α subunit.^8,9

The G-proteins have been classified into four major categories based on the sequence similarity of their Gα subunits: G_αs, G_αi/o, G_αq/11 and G_α12/13. (Fig.1) This classification defines both receptor specificity, as well as, in most cases, effector specificity.^5,6 The G_s and G_q have two well-defined effector pathways, the adenylyl cyclase (AC) and the phospholipase C-β (PLCβ) pathway, respectively.⁶ The activation pathways of the G_i and G_o are much less defined.

For many years researchers focused on the role that the different Gα subunits play in GPCRs activation pathways. However, today it is well established that the Gβγ subunit also mediates signal transduction by interacting with different effectors including AC, PI3 kinase, proteins of the MAPK pathway, and several ion channels.^5,6 (Fig. 4)

Ion channels are essential for the function of excitable cells by mediating electrical currents and controlling specific ion concentrations; however, they are also widely expressed in non-excitable cells.

Ion channels comprise a large family of transmembrane proteins. They regulate the movement of ions across the cellular membrane and can be divided into groups according to their ion specificity: Na⁺ channels, Ca²⁺channels, K⁺ channels, Cl^– channels and nonspecific cation channels.^10,11 Most ion channels are multisubunit proteins that undergo post-translational modification processes.¹²

GPCRs can modulate ion channel activity through two distinct pathways:

1. An indirect pathway that involves a common second messenger leading to the phosphorylation of the channel.

2. A direct pathway, involving binding of Gβγ directly to the channel also known as membrane delimited modulation.^13-16

A mechanism has been proposed which tries to explain the striking specificity of the signaling pathways, in spite of GPCRs diversity and the diversity of their effectors. This mechanism suggests the existence of a pre-assembled macromolecular signaling complex that targets a specific GPCR toward its ultimate effector. Such a complex was proposed for the β₂ adrenergic receptor and the L-type Ca_v1.2 channel¹⁷ and between dopamine receptors (D2 and D4) and the inward rectifier K_ir3 family and it seems to be relevant to other ion channels as well.¹⁸

Ion Channels Modulated by GPCRs

Ca²⁺ Channels

Ca²⁺ ions regulate many cellular processes such as secretion, proliferation, and apoptosis and are cofactors of many proteins.^19,20 In neurons, Ca²⁺ entry through Ca_v channels triggers neurotransmitter release. Fast synaptic transmission in the mammalian central nervous system (CNS) is mediated by several highvoltage-dependent Ca²⁺ channels.²¹ Five types of Ca²⁺ channels are expressed in the CNS: the L-type (Ca_v1), N-type (Ca_v2.2), P/Q-type (Ca_v2.1), R-type (Ca_v2.3), and the T-type (Ca_v3).¹⁶ Each Cav channel is a multimeric protein composed of a pore forming α1 subunit and the auxiliary β (Ca_Vβ), α2δ and γ subunits. There are four known Ca_Vβ subunits, in addition to four α2δ subunits and eight γ subunits. The best characterized Ca²⁺channels that are regulated by GPCRs are the Ntype and the P/Q-type which have significant roles in neuronal communication.²² This mechanism is the basis of synaptic modulation caused both by endogenous hormones as well as exogenously applied agents (such as analgesia caused by morphine). The identification of the types of Ca²⁺ channels that are modulated by GPCRs was enabled by the use of specific toxins: ω-Conotoxin GVIA for the N-type channels and ω-Agatoxin-IVA for the P/Q-type channels.²³

N-type and P/Q-type Ca²⁺ channels are negatively regulated by many different GPCRs by the two distinct mechanisms mentioned above. The indirect mechanism is voltage-independent and activates multiple intracellular signaling pathways, most of them involving second messenger and channel phosphorylation. Cell type, location of the channel within the neuron, and the type of neurotransmitter determines which G-protein subunit, G_αq, G_αi and/or G_βγ mediates the response.¹³ The second mechanism is a voltage dependent pathway. It inhibits the N-type and P/Q-type Ca²⁺ channels (but not the L-type) and involves direct interaction of the G_βγ with the α1 subunit. The degree of inhibition is dependent on both the combination of the G_βγ subunits and the type of channel.²⁴

A binding site for G_βγ that was demonstrated to be crucial for the voltage dependent inhibitory mechanism was found in the I-II linker domain (L1). The G_βγ binding site is adjacent to and slightly overlaps the binding site for the Ca_vβ of the channels and might give some clue as to the mechanism by which G_βγ exerts its inhibitory effect.^13,22,25 However, additional binding sites for the G_βγ subunit were found:

1. At the C-terminus of the α1 subunit, another G_βγbinding site that also overlaps with a Ca_vβ binding site, has been identified.^13,22,25

2. At the N-terminus where a putative binding sequence for the G_βγwas identified and was found to be critical for Ca_v2 voltage dependent inhibition.^13,26,27

Although three potential binding sites were described (Fig. 2), indirect kinetic studies pointed to only a single G_βγ binding site per channel raising the possibility that a combination of different G_βγ binding segments on the α1 subunit create the actual binding site for the G_βγ.¹³

K⁺ Channels

There are four known K⁺ channel families:

1. The voltage dependent K⁺channels designated as K_vchannels, which consist of twelve subfamilies.

2. The two pore domain channels, the K_2P, which consist of fourteen subfamilies.

3. The calcium activated K⁺channels, K_Cachannels, which consist of five subfamilies.

4. The inward rectifier K⁺channels, the K_ir, which include seven subfamilies, designated K_ir1 – K_ir7 with fifteen members.

GPCRs modulate a number of K⁺ channels. However, the most intensively studied and characterized are the K⁺ inward rectifier K_ir3 subfamily (K_ir3.1- K_ir3.4). A low basal level of activity, which is dramatically increased following stimulation of G_i/o-coupled receptors characterizes the K_ir3 channels.^28,29

Inward rectifier K⁺ channels (K_ir3, formerly named the GIRK channels: G-protein Inward Rectifier K⁺ channels), play an important role in maintaining the resting membrane potential by regulating the action potential duration and membrane excitability.³⁰ A functional K_ir3 channel consists of four subunits, which can be either identical, forming a homotetrameric channel or different, forming a heterotetrameric channel. The latter is the most abundant form of functional channel throughout the body.³¹ Different combinations of K_ir3 subunits have been demonstrated in different tissues. The cardiac channels are composed of K_ir3.1 and K_ir3.4 and are involved in regulation of cardiac rate. Neuronal channels are mainly composed of heterotetramers of K_ir3.1/K_ir3.2.^28,31,32

The K_ir3 channels are activated by various GPCRs that interact selectively with pertussis toxin (PTX)- sensitive Gαi/o proteins.⁹ They are activated when G_βγ subunits bind directly to the N- and C-terminal regions of each subunit. The channel can also be activated by phosphatidylinositol biphosphate (PIP2) which is essential for the proper gating of the channel following G_βγ activation.33 RGS proteins are responsible for the fast deactivation of the K_ir3 channels. The location of the four G_βγ binding sites per K_ir3 channel have been suggested to be at both the N- and the C-terminal regions. A synergistic enhancement between these four regions was suggested as a result of the close physical association of these regions.^7,34 Among the GPCRs that have been reported to modulate K_ir3 channels are the muscarinic receptors, P2Y receptors, somatostatin receptors, dopamine receptors and the GABA(B) receptors.

GPCRs Involved in Ca²⁺ and K⁺ Channel Modulation

As mentioned previously, a broad spectrum of GPCRs modulates ion channel activity. Adrenaline, glutamate, serotonin, acetylcholine (muscarinic), dopamine, opioids, P2Y, cannabinoid, somatostatin, and the GABA(B) receptors have all been shown to modulate Ca²⁺ and K⁺ channel actvity.^13-16,35 Several of these will be briefly reviewed below.

The Muscarinic Receptors

Muscarinic receptors are metabotropic acetylcholine receptors, and are widely distributed throughout the human body. In the CNS, there is evidence that muscarinic receptors are involved in motor control, temperature regulation, cardiovascular regulation, learning and memory.³⁶ There are five known muscarinic receptors: m1-m5. All five receptors are expressed in the brain, and each with a different distribution pattern. Muscarinic receptors can modulate both Ca²⁺ and K⁺ channels.

The muscarinic signaling pathway appears to be the most complex, and includes a large number of components such as: G-proteins, enzymes, second messengers, accessory proteins, cellular growth factors, transformation factors and ion channels.²⁹ Muscarinic receptors modulate Ca²⁺ channel inhibition through both mechanisms, the voltage independent and the voltage dependent. Accumulated data from pharmacological experiments demonstrated that in rat, as well as in mouse, the m1 receptor is involved in the voltage independent mechanism of inhibition, depressing several high voltage dependent Ca²⁺ currents.³⁷ The m1 receptor modulates both Ntype and L-type channels through the voltage independent mechanism involving a cytoplasmic second messenger.³⁷ This mechanism is related to muscarinic receptors m1, m3 and m5.

On the other hand, muscarinic receptors m2 and m4 are considered to mediate Ca²⁺ channel inhibition through the voltage dependent mechanism, by direct binding of the G_βγ subunit to the α1 subunit of the channel. Although both m2 and m4 are considered to modulate Ca_v channels by the same mechanism, a recent paper describes an experiment done with knockout mice which revealed that in m4 deficient mice the voltage dependent mechanism was not affected, while m2 knockout mice lacked the voltage dependent pathway.³⁷

Although the general mechanisms described above are well established, in different tissues and cell lines different types of muscarinic receptors participate in the activation of different pathways.

In NIH-3T3 cells, m1 activation did not affect the Ca_v3 (T-type channel) whereas activation of m3 and m5 receptors enhanced channel activity. m2 receptor affected channel activity only after elevation of cAMP levels by pre-treating the cells with Forskolin, resulting in the inhibition of the channel.

In HEK293 cells, expressing Ca_v2.3 channels, the R-type channel was shown to be both facilitated and inhibited by m1 and m2 receptors. However, inhibition was much weaker than facilitation for the m1 receptor, while for m2 receptors, inhibition was much more pronounced than activation.²⁹ The differential effects of muscarinic receptors on the R-type channel expressed in several cell lines, indicate that there might be differences in the susceptibility of the channel to modulation by muscarinic receptors. Facilitation and inhibition by the same receptors was probably exerted via different pathways as was indicated by their response to various inhibitors.²⁹ This might be correct with respect to other Ca²⁺ channels as well.

Muscarinic receptors, as mentioned before, also modulate K⁺ channels. Stimulation of muscarinic receptor m2 by acetylcholine in the heart results in activation of the K_ir3 channels through the membrane delimited mechanism. The G_βγ subunits activates the K_ir3.1/K_ir3.4 channels by directly binding to their N- and C-termini, causing a slower heartbeat.

Stimulation of m2 receptor by carbachol, in Xenopus oocytes injected with mRNA of K_ir3.1, K_ir3.4 and m2, activated K_ir3 channels. Activation was reversed by the use of Tertiapin, a specific blocker for Kir channels (which blocks both the K_ir3.1 and K_ir3.4 channels).³⁸ (Fig. 3,4)

m1, which is a G_q/11 coupled receptor, was shown to inhibit both basal and receptoractivated K_ir3 currents.^28,29

The exact mechanism is not clear. However, since activation of K_ir3 channels occurs through direct binding of the Gβγ to the channel, it was suggested that m1 inhibition occurs either by inactivating the G_i protein or perhaps by directly targeting the K_ir3 channel.²⁸

m1 receptor, in sympathetic neurons, was also found to inhibit the K_v7 (KCNQ) family (M-type K⁺ channels), which might be a result of local depletion in PIP2 which is needed in order to keep the channel in its open state.³⁹

The P2Y Receptors

Extracellular nucleotides such as ATP and UTP are usually co-released with other transmitters from cells and interact with cell surface P2Y receptors to produce a broad range of physiological responses: cardiac function, platelet aggregation and smooth muscle cell proliferation.⁴⁰

Eight mammalian P2Y receptors are known: the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14. Two subfamilies are defined within the P2Y family, which are distinct from each other on the basis of sequence identities and signaling properties:

1. The P2Y1 subfamily includes the P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors and is coupled to phospholipase C, mediating increases in inositol triphosphate (IP₃).

2. The P2Y12 subfamily consists of the P2Y12, P2Y13, and the P2Y14 receptors and is coupled to inhibition of AC.

All P2Y receptor subtypes are expressed in brain tissue and all are capable of modulating ion channels (with the exception of P2Y14 that has not been characterized).

In primary sympathetic neurons, P2Y1, P2Y2, P2Y4 and P2Y6 were all capable of inhibiting both Ca²⁺ and K⁺currents. Ca²⁺ channel (N-type) inhibition is achieved by both mechanisms: the voltage dependent and the voltage independent. Inhibition of K⁺ channels (K_v7.2, 3 and 5) by those receptors is mediated by PLC and IP₃-dependent increases in intracellular Ca²⁺.^41,42 Although P2Y1, P2Y2 and P2Y6 receptors were shown to equally inhibit both Ca²⁺ and K⁺ current, coupling of the P2Y4 receptor to K_V7 channels was much more efficient than to Ca²⁺ channels.⁴² The P2Y12 receptor, in contrast, inhibits only Ca²⁺ channels through the voltage dependent mechanism.^41,43,44 It seems that the P2Y13 receptor, like P2Y12, also inhibits N-type Ca²⁺channel via the G_βγ subunit possibly, through the voltage dependent mechanism, however, this is not well established.⁴⁵

The Somatostatin Receptors

Somatostatin is a small cyclic peptide that is widely expressed throughout the CNS and peripheral tissues.⁴⁶ In peripheral tissues, somatostatin exerts inhibitory effects on secretion processes, whereas in the brain it acts as a neurotransmitter in both a stimulatory and inhibitory manner.^45,46 Six receptors, encoded by five genes, underlie the action of somatostatin: SSTR1, SSTR2 (with two known alternative spliced isoforms; SSTR2a, SSTR2b), SSTR3, SSTR4 and SSTR5.^46,47 All somatostatin receptors are expressed in the CNS, and in endocrine/exocrine glands.⁴⁸

Activation of somatostatin receptors is coupled to multiple signaling pathways. All somatostatin receptors inhibit AC activity while at the same time are able to selectively activate other transduction targets.⁴⁸ SSTRs also modulate several ion channels. Among them are the K_ir channels, K_v channels, Ca²⁺ activated K⁺ channel (K_Ca) and the high voltage activated L- and N-type Ca_v channels.^48,49 However, inconsistent results were reported with SSTR1 and SSTR2 in heterologous systems in respect to their coupling to AC and to ion channels. In pituitary cells, somatostatin receptor was found to activate K⁺ channels and to inhibit voltage dependent Ca²⁺ channels.⁵⁰ In the pancreatic β-cell line MIN-6, SSTRs activated two types of inward rectifier K⁺ channels; the KATP and K_ir3 channels.⁵¹ Somatostatin receptors were also shown to modulate the transient receptor potential vanilloid 1 channel (TRPV1).⁵² DRG cells expressing TRPV1 also express, in most cases, SSTR2.

In vivo behavioral studies indicated that SSTRs can modulate the TRPV1 channel and this is further supported by in vitro experiments.⁵²

The Cannabinoid Receptors

Cannabinoids have been used in eastern medicine for many years as pain relievers.⁵³ To date, two specific cannabinoid receptors, known as CB1 and CB2, have been identified. Both of them are coupled to the G_i/o G-protein.^54,55 CB1 was shown to modulate Ca²⁺ and K⁺ ion channels in different tissues and cell lines.^54-57Inhibition of pre-synaptic N-type and P/Q-type Ca²⁺ channels was demonstrated by several cannabinoids (endocannabinoids as well as exogenously applied) in heterologously expressed mammalian neurons,⁵⁵cultured hippocampal neurons,^57,58 neuroblastoma-glioma cells (N-type only) and in exogenously expressed CB1 receptor in pituitary tumor cells (P/Q-type).⁵⁹ Endocannabinoid stimulation of the CB1 receptor was also shown to activate the K_ir3 channels both in pituitary tumor cells and in heterologously expressed mammalian neurons, where it was shown that ion channel modulation by the CB1 receptor is also dependent on expression levels of the receptor.⁵⁵ It has been suggested that different agonists of CB1 receptor might affect the selectivity of the interaction between the receptor and the G-proteins by inducing different conformational states of receptor.⁶⁰

The GABA(B) Receptors

GABA(B) receptors mediate slow synaptic inhibition in the brain and spinal cord and are activated by gamma aminobutiric acid (GABA) which is a major inhibitory neurotransmitter.

GABA(B) receptors are coupled to the pertussis toxin-sensitive G-proteins, the Gα_i/o.^61,62 The functional GABA(B) receptor is a heterodimer, which consist of two subunits, GABA(B) R1 and GABA(B) R2. Cell surface expression is also dependent on the receptor being a heterodimer.^61,62 GABA(B) R2 was shown to be essential for trafficking of the receptor to the cell surface as well as for activation of effector systems, while GABA(B) R1 was found to be important for the ligand binding along with GABA(B) R2.^63-65 Inhibition of Ca²⁺ (N-type and P/Q-type) channels on one hand and on the other hand activation of K_ir3 channels by GABA(B) receptors has been demonstrated, probably by the same voltage dependent mechanism involving direct binding of the G_βγ subunits.^66,67

Summary Remarks

Ion channel modulation by GPCRs is much more diverse and complex than what was first expected. The diversity of ion channels along with the large number of GPCRs, and G-protein combinations creates a diverse array of signaling pathways which control cellular functions. The complexity is growing while new functions are constantly being discovered, unmasking known orphan GPCRs. Both ion channels and GPCRs have become targets in the search for therapeutic drugs in cancer, heart diseases, obesity among others.

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