GPCR and Voltage-Gated Calcium Channels (VGCC) Signaling Complexes - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 240

Lohse et al. 2007 ). Combination of these assays with electrophysiological record-
ings and immunocytochemistry to observe the cellular distribution and the trafficking
of these signaling complexes, has provided insights into the functional and physical
crosstalk between ion channels and receptors.

13.2.3.1 GPCR and L-Type Channel Complexes

Because of their importance in cardiac physiology, the regulation of L-type calcium
channels by b2-adrenergic receptor (b2-AR)-cAMP-PKA signaling pathway has
received intense attention. Cav1.2 encodes L-type channels in cardiac muscle cells
and these channels undergo phosphorylation and upregulation in response to b 2-AR
agonists (Skeberdis et al. 1997 ; Brodde and Michel 1999 ; Bodi et al. 2005 ) . Although
the enhancement of L-type channels activity has been largely reported in native
cardiac cells, the reconstitution in a heterologous expression system of such regula-
tion has been challenging and controversial until recently. While L-type current
density is increased by 200-300% in native cardiomyocytes (Hulme et al. 2003 ) and
neurons (Oliveria et al. 2007 ; Davare et al. 2001 ) stimulated with b 2-AR agonists,
the potentiation never reached more than 50 % in a recombinant system (Gao et al.
1997 ; Fraser et al. 1998 ). Interestingly, Cav1.2 channels and b2-AR are part of the
same macromolecular signaling complex and both proteins bind to the scaffold
A-Kinase Anchoring Protein (AKAP) which optimizes the coupling between the
b2-AR, PKA and the channel (Hall et al. 2007 ; Malbon 2007 ; Dai et al. 2009 ) .
AKAPs define a group of scaffolding proteins that display a signature binding site
for the RI/RII subunit of protein kinase A. They optimize ion channel phosphoryla-
tion by targeting the kinase to specific intracellular locations and consequently
provide spatial and temporal control over cAMP signaling events. AKAPs are
multivalent and interact with calcium regulating proteins in adult cardiomyocytes:
for example, AKAP7 (AKAP15/18) binds L-type channels (Gray et al. 1997 ) ,
AKAP6 (mAKAP) binds RyR2 ryanodine receptors (Marx et al. 2000 ) AKAP12
(gravin) associates with the bAR (Fan et al. 2001 ) , and fi nally AKAP5 (79/150)
(Fraser et al. 1998 ) can interact with multiple proteins, including the L-type channel
Cav1.2, b-AR, adenylyl cyclase, PKA, PKC, and calcineurin (protein phosphatase
2B, PP2B) (Fraser et al. 2000 ; Malbon et al. 2004 ; Bauman et al. 2006 ; Malbon
2007 ; Dai et al. 2009 ). A recent accomplishment from the Catterall group was to
accurately reconstitute this macromolecular signaling complex in a heterologous
expression system, which confirmed the necessity of partner molecules such as
AKAP (Fuller et al. 2010 ). They found that proteolytic cleavage of the distal
carboxy-terminal tail of the channel would lead to channel inhibition through intra-
molecular interaction of the proteolytic fragment with the proximal C-terminus of
Cav1.2. Furthermore, they demonstrated that a precise cDNA ratio of Cav1.2 to
AKAP15 (the PKA anchoring protein in cardiac cells) was required to observe a
magnitude of L-type channel modulation similar to that described in native cardio-
myocytes. The importance of the macromolecular signaling complex is not restricted
to an increase in L-type current density. In neuronal cells, the activation of transcription