Role o f P subunits

The P 1 and P2 subunits from rat brain are not closely related in terms of amino acid sequence, but each contains a single membrane spanning segment that separates a large extracellular amino terminal from a smaller intracellular carboxy terminal (Isom, et al 1992, 1995).

1.7.1.1. p i:

The p i accessory subunit is non-covalently associated with the a-subunit. Co­ expression of p i subunit with rat brain Navl.2 (type II) sodium channel or skeletal muscle (Navi .4) a-subunit in Xenopus oocytes increased the size o f the peak sodium current, accelerated its activation, and shifted the voltage dependence of inactivation to more negative potentials. This indicates that p i is crucial in the assembly, expression and functional modulation of the rat brain sodium channel heterotrimeric complex. (Isom et al, 1992; Smith & Goldin, 1996, 1998; Patton et al, 1994; Schreibmayer et al, 1994). Chen & Cannon (1995) showed through deletion analysis that the intracellular domain of human P1 is not required for modulation of the skeletal muscle a-subunit, whereas the extracellular domain is sensitive to deletion mutagenesis. In addition, the P 1 extracellular domain together with proximal residues of transmembrane domain, was found to be sufficient for modulation of the skeletal muscle Na^ channel in chimeric subunits formed with p2 subunits (Makita et al,

1996). McCormick et al, 1998 demonstrated through deletion mutagenesis and chimeric protein analysis that the extracellular domain of p i forms an Ig fold that is essential for expression and function of this subunit. Co-expression in mammalian cells of Navl .2 (SCN2a) a-subunit and p i increased the level of sodium channels at the plasma membrane two- to four fold (Isom et al, 1995) showing that p i subunit

expression is a critical regulator of sodium channel density in the plasma membrane of transfected cells, p i does not appear to modulate all sodium channel a-subunits. This is seen from the observation that N avi.8 (SNS) a-subunits are not affected by co­ expression of p i (Sangameswaran et al, 1996). These results suggest that other pi-like subunits may be present in sensory neurones, p i i s only expressed after birth in the developing brain (Patton et al, 1994). From developmental time course studies, p i expression in rat forebrain showed multiple size bands at earlier time points; these bands were also present in adrenal glands, heart and skeletal muscle. Qu et al (1999) demonstrated through studies with chimeras that segment IVSS2-S6 of the a subunit played an important role in modulation of gating by p i. They proposed that this segment may be one site of interaction for the P 1 molecule.

I.7.I.2. piA :

In an attempt to identify other pi-subunit isoforms a rat adrenal cDNA library was screened from which a cDNA clone was isolated that had identity to the 5' region of pi followed by a novel 3' end. This p i isoform, piA , was a splice variant of pi (Kazen-Gillespie et al, 2000). The developmental time course of p i vs. p iA mRNA expression in rat brain showed p i A is expressed early in embryonic development. Its expression declines to undetectable levels after birth, concomitant with the expression of p i. Immunohistochemical analysis of p iA expression revealed that it is expressed in adult DRG, spinal cord and heart. Functional co-expression of Navi .2 (SCN2a) with p i A in transfected Chinese hamster lung fibroblast resulted in a 2.5 fold increase in current density compared with cells expressing a-subunit alone. This increase in current density reflected two distinct effects of piA , one is the increase in the

proportion of cells expressing detectable sodium currents and secondly an increase in the level o f functional sodium channels expressing cells (Karen-Gillespie et al, 2000). These increases in N a v i.2 sodium channel expression in mammalian cells with p i A are similar to the ones obtained for p i (Isom et al, 1995) and in Xenopus oocytes

(Isom et al, 1992).

1.7.1.3 P2:

Partial proteolytic maps showed that p i and P2 subunits of brain Na"^ channel are distinct and unrelated in terms of amino acid sequences (Messner & Catterall, 1985). P2 is associated to the a-subunit via a disulphide link (Hartshorne et al, 1982). Sequence analysis of P2 revealed that its extracellular domain contains an immunoglobulin like fold and an extended region with similarity to cell adhesion molecule contactin (Isom et al, 1995).

Co-expression of P2 with a-subunits in Xenopus oocytes caused an increase in functional expression of sodium channels, an increase in the fraction of a-subunits gating in a fast mode, and a small negative shift in the voltage dependence of channel inactivation similar to the effects observed with p i (Isom et al, 1995). Expression of higher levels o f P2 also caused a four fold increase in the capacitance of Xenopus

oocytes, which resulted primarily from an increase in the number and surface area of the plasma membrane microvilli. This p2 mediated increase in membrane capacitance did not depend on co-expression with the a-subunit. p2 is not required for Na^ channel function as it is only found in neuronal Na^ channels and not in muscle. The sequence similarity of P2 subunits to contactin, their ability to expand the cell surface

membrane, and their appearance in developing neurones and axons suggested that they may modulate cell surface expression and function of sodium channels during neurogenesis and synaptogenesis (Isom et al, 1995).

I.7 .I.4 . |33:

Morgan et al, (2000) have identified a rat and human form o f p3 which is closely related to p i but exhibits a complementary distribution in the rat central nervous system. The coding region of rat p3 nucleotide sequence is 57% identical to rat p i and 40% identical to rat p2 and is a separate gene product. As with p i and P2, P3 has an extracellular domain which shows homology to proteins that adopt a V-type Ig fold (Harpaz & Chothia, 1994). Of these proteins, the extracellular domain of p3 has the highest sequence identity to myelin Po (Shapiro, 1996). The known structure of myelin ?o was used as a template to model P3 and thus identify regions of functional importance and facilitate a comparison with the previously predicted p i structure (McCormick et al, 1998). When co-expressed in Xenopus oocytes with rat Navl.2 a - subunit, p3 causes slower inactivation of Na^ channel opening than p i (Morgan et al,

2000).