GIRK Revision

Question 1

How do GPCR’s bind ligands?

Answer 1

usually by capturing the ligand with the N-terminus and/or with a pocket formed by the extracellular loops of the transmembrane domains

Question 2

How does ligand binding generate a signal transduction?

Answer 2

Ligand binding generates a signal transduction response through activation of specific intracellular GTP binding proteins (G proteins). GPCR’s may be located in the synapse or peri-synaptically allowing for the mediation of slow synaptic transmission (time scale of 100ms to seconds); whose characteristics will depend on the particular G-protein that couples to the receptor

Question 3

G proteins are heterotrimeric proteins composed of

Answer 3

alpha, beta and gamma subunits which act as transducers between the receptor and effector system

Question 4

What modifications do G proteins have and who showed this?

Answer 4

G alpha proteins are typically anchored to the membrane by a N-terminal palmosylation or mitrosylation (Vogler, 2008). While Ggamma subunits are isoprenylated at the CAAX motif in the C-terminal (Higgens and Cassey, 1994). In contrast, G beta subunits have no membrane-anchoring post-modificational changes rather, they bind tightly to the gamma subunit through hydrophobic interactions (Sondek, 1996).

Question 5

What do Go/i proteins do?

Answer 5

Gi inhibits AC while Go inhibits voltage-gated Ca2+ and activates K+ channels. Both are inhibited by petruses toxin (PTX)

Question 6

What do G12/13 proteins do?

Answer 6

G12/13 stimulates PLC-epsilon and RhoA and is insensitive to cholera toxin and PTX

Question 7

Describe the GTP binding domain?

Answer 7

The GTP binding domain is homologous to Ras-like small GTPases, and includes switch regions I and II, which change conformation during activation.

Question 8

How do GPCR’s elicit signalling?

Answer 8

Upon GTP binding, a conformational change in Galpha leads to its separation from the G beta-gamma dimer, so that the subunits can interact with downstream targets.

Question 9

How is GPCR signalling terminated?

Answer 9

The intrinsic GTPase activity of the Gα subunit hydrolyzes bound GTP back into GDP + Pi, thus terminating Gα activity and promoting the reassembly of the inactive Gα-GDPGβγ complex.

Question 10

What influences GTPase activity?

Answer 10

GTPase activity can be accelerated by a family of RGS proteins (regulator of G protein signalling) which thus influence the extent and duration of downstream events.

Question 11

Under physiological conditions, the small outwards

Answer 11

K+ current allows for the inhibition and regulation of neuronal excitability

Question 12

The first studies of the actions of heterotrimeric G proteins on GIRKS came from measurement of the cardiac GIRK current (IkACh). First evidence of a novel signalling pathway came from

Answer 12

Seojma and Noma, 1984, who used cell-attached patch clamp recordings of rabbit atrial cells to demonstrate that Ach applied through a bath solution showed no effect on GIRK’s but Ach applied through a pipette enhanced GIRK activity. This suggested that G-proteins produce a membrane-delimited activation on GIRK channels that was not dependent on the activity of cytoplasmic second messengers.

Question 13

What study suggested that the membrane delimited effects of GPCR’s on GIRK’s was due to Galpha?

Answer 13

purified native or recombinant Ga subunits activated by GTPyS (non-hydrolysable) on inside-out patches was found to enhance GIRK activity which suggested that the Ga subunit was responsible for this response (Yatoni,1988)

Question 14

What initial study suggested that it was Gby not Ga that acted on GIRK’s?

Answer 14

Logotethis et al.,1987 found that application of purified native or recombinant Gby and not Ga mediated GIRK activity in rat atrial cells

Question 15

What allowed 2 studies to be conducted in 1994 that showed Gby activated GIRK’s?

Answer 15

cloning of GIRK subunits and improved protein expression systems and purification techniques

Question 16

What were two studies in 1994 that showed Gby-dependent activation of GIRK’s?

Answer 16

expression of recombinant Gβγ subunits led to sustained activation of GIRK channels, which was attenuated by a Gβγ binding protein, the β-adrenergic receptor kinase (βARK) (Reuveny et al., 1994).

A second study using inside-out patches of isolated rat atrial cells showed that different combinations of recombinant and purified Gβγ dimers, but not Gα, in the presence of GTPγS (thus preventing the reassociation of Gβγ with Gα) directly activated GIRK channels (Wickman et al., 1994).

Question 17

What role does Galpha play in Gby-GIRK modulation?

Answer 17

PTX-sensitive Gai and Gao are crucial regulators of Gby effects on GIRK due to their close proximity and high affinity binding of Gby to form Gaby which controls basal channel activity and expression.

Question 18

What is the Gby binding site on GIRK’s formed from?

Answer 18

the interface of two subunits in the outer side of the cytosolic terminal domain (CTD). The interface is formed of sequences from the N-terminal domain and the BD-BE and BL-BM sheets of the CTD.

Question 19

What conformational change on GIRK’s does Gby lead to?

Answer 19

conformational change of the BL-BM sheets which leads the NTD and slide loops to transmit the signal to the M2 and G-loops near the membrane which stabilizes open conformation of the channel.

Question 20

What was first described about VGCC’s 40 years ago?

Answer 20

G protein inhibition of voltage-gated calcium channel (VGCC) currents was first described over 40 years ago in two seminal articles by Dunlap and Fischbach

Question 21

Describe the VGCC structure

Answer 21

These channels consist of the main α1 subunit, that when folded makes up the ion pore, and 4 accessory subunits termed δ, γ, β and α2

Question 22

What did Dunlap and Fischbar show?

Answer 22

These authors showed that the contribution of VGCCs to somatic action potentials in chick dorsal root ganglion (DRG) neurons was reduced in response to activation of GABAB, serotonin, or adrenergic receptors, thus shortening action potential duration (Dunlap and Fischbach, 1978). Subsequently, they showed that this effect was due to a robust, receptor-mediated inhibition of high voltage activated (HVA) calcium currents in chick DRG neurons, currents now known to be carried almost entirely by N-type channels (Dunlap and Fischbach, 1981)

Question 23

What did studies about out GPCR’s on VGCC’s show?

Answer 23

Numerous studies followed, revealing that many types of GPCRs—muscarinic, opioid, somatostatin, and dopamine receptors among them— have the propensity to inhibit native calcium currents

Question 24

What was a unifying property of GPCR-mediated inhibition of calcium currents?

Answer 24

A unifying property of this GPCR-mediated inhibition of calcium currents was its sensitivity to pertussis toxin, thus implicating Gαi and/or Gαo proteins (Holz et al., 1986).

Question 25

What implicated Ga subunits in VGCC GPCR inhibition?

Answer 25

The receptor-mediated inhibition was found to be blocked by the application of GDPβS (Holz et al., 1986), further implicating Gα subunits.

Question 26

Has a role in Ga in VGCC inhibition been found?

Answer 26

A role for Gα subunits in this mode of inhibition was thus established, yet not well understood.

Question 27

What implicated membrane proteins in VGCC GPCR block (and how does the relate to Gby?

Answer 27

The inhibition was then found to be membrane-delimited (Forscher et al., 1986). Because the Gβγ subunit is anchored to the plasma membrane, these findings were consistent with direct, inhibitory physical interaction between VGCCs and membrane-tethered Gβγ subunits

Question 28

Which papers implicated a direct role for Gby?

Answer 28

A direct role for Gβγ in the inhibition of VGCCs was first proposed by Bourinet et al. (1996) and later demonstrated in experiments testing the effects of overexpressed Gβγ subunits. The results of these experiments were fully consistent with the notion of a membrane-delimited pathway and were reported in back-to-back publications by the Ikeda and Hille groups in 1996.

Question 29

In what way is Gby block of VGCC’s subtype dependent?

Answer 29

N-type channels have long been considered a prime target for direct G protein inhibition, and it is now established that P/Q-type VGCCs are also inhibited upon activation of Gαi- or Gαo-coupled receptors. However, P/Q-type channels typically undergo a smaller degree of inhibition relative to N-type channels (Currie and Fox, 1997). In contrast, other subtypes expressed in native cells do not seem to be subject to direct G protein inhibition, suggesting that this type of modulation is confined to the two main presynaptic calcium channel isoforms.

Question 30

What do we know about Gby blockage of VGCC’s?

Answer 30

The mechanism by which bound Gβγ prevents the channel from opening is unknown. In one study, the effect of G protein activation on voltage sensor movement in CaV2.2 was examined. This revealed that GTPγS produced a depolarizing shift in the voltage dependence of charge movement that could be reversed by a large depolarizing prepulse and also induced the appearance of a slow component of “on” gating charge. The greatest effect was the large separation on the voltage axis between gating charge movement and channel opening. Thus, Gβγ is acting both to slow voltage sensor movement and to inhibit the subsequent transduction of this movement into channel opening (Jones et al., 1997).

Question 31

Why has the N terminal been implicated in Gby binding of VGCC’s?

Answer 31

Several studies have attributed a key role to the N terminus of the channel in G protein inhibition. In particular, three individual amino acid residues in the Cav2.2 N terminus (Ser48, Arg52, and Arg54) were found to be critical for N-type VGCC modulation (Canti et al., 1999).

Question 32

Why might the beta subunit be important for Gby binding to VGCC’s?

Answer 32

Agler et al. (2005) showed that the N terminus of Cav2.2 and the I-II linker, which partially overlaps with the AID region that is known to bind the calcium channel β subunit, physically interact with each other, and this interaction is essential for G protein modulation to proceed. This overlap raises the question of whether Gβγ could bind to the I-II linker in the presence of the β subunit, either forming a stable quaternary complex with the α1 and β subunits or a scenario of mutually exclusive binding of either the Gβγ heterodimer or the calcium channel β subunit to the α1 subunit (Figure 5). Regardless, the β subunit is an important determinant of G protein inhibition of Cav2 calcium channels.