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CaN has an autoinhibitory (Al) domain whose function is to block the catalytic site and prevent enzyme activity. The binding of CaM is thought to displace this domain and cause an increase in Vmax, without altering Km (Klee et al., 1988). CaN can also be activated by limited proteolysis, and removal of a third of the C- terminus still yields a fully active enzyme. Interestingly, proteolysis in the presence of Ca^VCaM yields an constitutively active enzyme that is no longer sensitive to Ca^VCaM. Investigation of the Al domain by Hashimoto et al., (1990), revealed a 25 amino acid sequence (corresponding to residues 467-492; Fig.1.5) from the C-terminus which could inhibit the enzyme with an IC50 of 10-15 pM

(Hashimoto et al., 1990). This peptide known as calcineurin autoinhibitory peptide (CAP) has been shown to inhibit CaN activity in a variety of in vitro assays (Perrino, 1999). The Al domain also contains a consensus motif for casein kinase II, and whilst CaN is a substrate for this enzyme in vitro, it is.not clear if it is in vivo (Guerini and Klee, 1989).

1.4.3 Inhibition by cyclosporin A and FK-506

CaN achieved clinical significance when it was discovered that the immunosuppressants, cyclosporin A and FK-506 act via the inhibition of CaN. Unlike most other drugs, these agents must first bind to an intracellular ligand, known as an immunophilin, in order to have an inhibitory effect. Immunophilins are peptidylprolylisomerases, although their function in physiological CaN signalling is unclear. Immunophilins are known to form complexes with the sarcoplasmic reticulum ryanodine receptor (Lam et al., 1995; Bandyopadhyay et al., 2000), and the IP3 receptor (Cameron et al., 1995) and thus may be involved

in Ca^^ signalling. Cyclosporin A is known to bind to the immunophilin, cyclophilin, whilst FK-506 binds to the FK-506 binding protein 12 (FKBP-12) (Liu et al., 1992). The direct mechanism of inhibition is unclear. Whilst the Al domain of CaN inhibits activity against all known substrates, cyclosporin A and FK-506 do not, suggesting that the binding site for the immunophilin complex may be distinct from the catalytic site, and may in fact be on the CnB subunit (Hashimoto et al.,

1990).

1.4.4 Regulation of Protein Phosphatase 1

PP-1 can be inhibited by the endogenous phosphoprotein, inhibitor-1 (1-1). 1-1 can be reversibly phosphorylated (and activated) by PKA. Importantly, 1-1 is also a substrate for CaN (Klee et al., 1988), and thus CaN can also regulate the activity of Ca^^-independent phosphatases. Dephosphorylation of 1-1 by CaN, inactivates the protein resulting in activation of PP-1. In this way, PKA might act not only by phosphorylating its cellular target, but also by inactivating an inhibitory mechanism (phosphorylation of 1-1). The observation that PKA itself is a

substrate for CaN merely adds yet another layer of complexity to this pathway. Determining whether an effect is mediated by CaN directly, or indirectly, via PP-1, can be elucidated using selective inhibitors of PP-1 and PP-2A. An effect that is sensitive to cyclosporin A or FK-506 as well as calyculin A would infer the latter pathway, whilst an effect sensitive to cyclosporin A or FK-506 and insensitive to calyculin A would suggest a direct CaN effect (Perrino and Soderling, 1998).

1.4.5 Regulation of ion channeis by caicineurin

A role for caicineurin in modulation of ion channel activity has largely come through the use of the caicineurin inhibitors, cyclosporin A and FK-506., Both Na^, Ca^* and K* channels, as well as the NaVK^ ATPase have been found to be sensitive to the effects of these agents (Perrino and Soderling, 1998). In smooth muscle however, only Ca^* channels have so far been reported to be regulated by CaN. It is long established that L-type Ca^^ channels are activated by PKA and inhibited by Ca^^ (Hadley and Lederer, 1991), the effect of Ca^^, clearly being a negative feedback mechanism. The molecular nature of this inhibition was recently elucidated when it was demonstrated that application of purified CaN to the cytosolic face of an excised patch of umbilical vein smooth muscle cell, caused inactivation of Ca^^ channels (Schuhmann et al., 1997). Up until now, CaN has not been implicated in the regulation of any mammalian K* channel.

1.5 Physiological and pathophysiological roles of NO 1.5.1 Nitric oxide and K* channels

Since the discovery that NO mediates many of the vascular effects attributed to endothelium-derived relaxing factor (EDRF; (Moncada et al., 1991)), a substantial

number of studies have sought to determine the mechanisms by which NO elicits vasorelaxation. Vasorelaxation induced by the NO pathway is ultimately achieved by a variety of mechanisms, the contribution of which varies between tissues and species. Because NO itself can elicit membrane hyperpolarisation and relaxation in several blood vessels (Cohen et al., 1997), channels are a likely target for mediating at least some of the effects of NO. In particular, there is considerable evidence to suggest that BKca channels can be activated by NO either directly,

via nitrosylation of channel proteins (Fig. 1.7) (Bolotina et al., 1994; Mistry and Garland, 1998; Abderrahmane et al., 1998b), or indirectly, via PKG mediated phosphorylation of channel residues (Robertson et al., 1993; Abderrgjimane et al., 1995). Furthermore, the Kca channel inhibitors, ChT) and also IbTx are effective at reversing part of the relaxation induced by NO and NO donors in a number of blood vessels (Archer et al., 1994; Bialecki and Stinson-Fisher, 1995; Plane et al., 1998). In other studies, both Katp (Murphy and Brayden, 1995) and

Kv channels (Yuan et al., 1996) have been implicated in mediating the

hyperpolarisation to NO in mesenteric and pulmonary artery, respectively. Taken together, these observations imply that NO can activate more than one type of channel, and that the target varies between vascular beds. However, many studies investigating the role of smooth muscle K* channels in NO-induced relaxation have been performed in the presence of the endothelium, which itself expresses a variety of Ca^^-dependent channels (Marchenko and Sage, 1996) that may be sensitive to NO. Therefore, some of the effects of K* channel inhibitors may be related to an effect on the endothelium rather than the smooth muscle (Edwards et al., 1998).

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