Functional properties

Functionally, the presence of VDCC p subunits in expression system studies has revealed a repertoire of effects on the m ajor a i subunit. These are sum m arised below.

1.2.2.3.2.1 p subunit is obligatory for surface expression and correct functional assembly of VDCCs

The P subunit is thought to act as a chaperone to traffic V D CCs to the cell m embrane. Several groups have investigated this property. To determ ine the role o f the pi subunit in channel activity and excitation-contraction coupling, G regg et a l (1996) ) used gene targeting to inactivate the Pi subunit in mice. These m ice died at birth from asphyxia resulting from a lack o f excitation-contraction coupling. Im m unohistochem istry o f cultured myotubes show ed that not only was the Pi subunit absent, but the am ount of Cay 1.1 in the m em brane was also undetectable. Im m unocytochem istry in Cay 11 null m yotubes reveal that pia expression is normal and appropriately localised. Gregg et a l therefore concluded that the pi subunit plays an im portant role in both the transport and insertion of the Cay 1.1 subunit into the m em brane and the targeting of the muscle D H PR com plex to its appropriate functional location (G regg et a l, 1996). Supporting evidence for the chaperoning role o f p subunits cam e from several other sources; the rescue of excitation-contraction coupling in pi null m yotubes following restoration of the Pi subunit expression (Beurg et a l ,

1997); the knockout o f endogenous Xenopus oocyte p subunits resulting in inhibition o f transient V DCC expression (Tareilus et a l , 1997); co-expression o f C ay 2 .1 with pib, p2a, or p3 resulted in channel targeting to the m em brane o f C O S -7 cells (Brice et a l ,

1997) and m oreover differential p subunit association controlled the sub-cellular localisation o f the V DCC com plex (Brice and D olphin, 1999); a point m utation in the A ID on the a i I-II linker prevented p subunit m ediated chaperoning o f Cay 1.2 to the cell m em brane (G erster et a l , 1999); the a i subunit I-II linker possesses an endoplasm ic reticulum retention signal that is m asked on interaction w ith a p subunit, facilitating insertion o f the channel into the plasm a m em brane (Bichet et a l , 2000). A lthough Cay3

Chapter 1__________________________________________________________Introduction

T-type channels possess no classical P subunit interaction dom ains, D olphin et a l

reported that Pib produced an increase o f functional expression o f Cav3.1, either in the absence or the presence of heterologously expressed a 2 ô , w hereas the other p subunits had m uch sm aller effects (Dolphin et a l , 1999b), though co-expression o f a p subunit was not obligatory for channel function.

The chaperoning properties of the p subunit are^ how ever^independent o f its allosteric m odulation o f the a ; subunit, which influences the activation and inactivation properties o f the channel (G erster et a l , 1999; Yamaguchi et a l , 1998).

1.2.2.3.2.2 Setting of proper kinetics of activation and voltage dependence of activation.

The currents expressed by Cay 1.1 subunits transfected alone in L-cells w ere extrem ely abnorm al and only resem bled native channels upon co-expression o f the p subunit (Lacerda et a l , 1991; Varadi et a l , 1991). Co-expression o f the pia subunit w ith the cardiac Cay 1.2 channel consistently enhanced expressed currents, accelerated activation kinetics and shifted the voltage-dependence of activation to hyperpolarized potentials (Singer et a l , 1991; W ei et a l , 1991). These properties are reproduced in the Cay2 fam ily o f channels (Canti et a l , 2000; De W aard and C am pbell, 1995; O lcese et a l,

« 1996; Stephens et a l , 2000). p subunits facilitate the opening o f the channels by im proving the coupling between channel opening and charge m ovem ent during a depolarisation (Neely et a l , 1993). This alteration m anifests as an increase in the steepness o f the conductance-voltage relationship, by increasing the open probablilty o f channels and hence a left-shift o f the half maximal value for voltage dependent activation (K am p et a l , 1996; O lcese et a l , 1996).

1.2.2.3.2.3 Inactivation

W hilst a i subunits contain the innate determ inants o f voltago-dependent inactivation (Cens et a l , 1999; H erlitze et a l , 1997; Spaetgens and Zam poni, 1999; Z hang et a l ,

1994), association with p subunit isoform s dictates their overall inactivation rate (De W aard and Cam pbell, 1995; Olcese et a l , 1994). In general, the differential association

Chapter 1__________________________________________________________Introduction

of a H V A tti subunit with a p subunit influences the rate o f voltage-dependent inactivation in the following order (fastest to slowest) p3 > pib ~ p4>> pia (De W aard and Cam pbell, 1995; Patil et al., 1998; Stea et al., 1994). The properties o f the p2a are in fact the opposite Of the other three p subunits, shifting the voltage dependence of inactivation m ore positive rather than m ore negative, and significantly slow ing the inactivation kinetics (Olcese et al., 1994; Qin et al., 1996; W yatt et al., 1998). This property is controlled by the differential splicing o f the N -term inus (O lcese et al.,

1994), w hich as previously described is unique in the p2a subunit, w ith additional contributions from the long form o f the D3 region o f the subunit (Qin et al.,

1996)(Figure 1.13 B- Region 3).

1.2.2.3.2.4 Prepulse facilitation

As previously described, the P subunit and the Gpy subunit appear to share com m on interaction motifs on the tt] subunit o f m em bers o f the Cav2 fam ily and that the P subunit reduces the m agnitude o f G -protein inhibition by enhancing the rate of dissociation o f the Gpy from the a j subunit during a depolarising prepulse (R oche et al.,

1995; R oche and Treistm an, 1998). This theory is supported by experim ental evidence where the P subunits were depleted by injection o f antisense nucleotides into dorsal root ganglion cells, resulting in increased Gpy m ediated inhibition (Cam pbell et al., 1995). H ow ever,sub sequ ent findings reported that, although varying the concentration o f p subunit alters G-protein modulation of VDCC calcium currents (Canti et al., 2001), when p subunit expression is com pletely absent, voltage -dependent facilitation of currents by prepulse is lost because the p subunit is no longer present to facilitate the rem oval o f bound Gpy subunits (M eir et al., 2000).

L-type channels also experience voltage-dependent facilitation, but although it is independent o f G -protein involvement, research suggests that co-expression o f the auxiliary p subunit is necessary (Bourinet et al., 1994; Kamp et al., 2000).

Chapter 1__________________________________________________________ Introduction