Three homologous genes, Cnn1, Cnn2 and Cnn3, have evolved in vertebrates, encoding three isoforms of calponin: calponin 1,^[3][4] calponin 2,^[5] calponin 3,^[6] respectively. Protein sequence alignment shows that calponin 1 is highly conserved in mammals but more diverged among lower vertebrates.

The expression of CNN1 is specific to differentiated mature smooth muscle cells, suggesting a role in contractile functions. Calponin 1 is up-regulated in smooth muscle tissues during postnatal development^[7] with a higher content in phasic smooth muscle of the digestive tract.^[8]

The majority of structure-function relationship studies of calponin were with experiments using chicken calponin 1. Primary structure of calponin consists of a conserved N-terminal calponin homology (CH) domain, a conserved middle region containing two actin-binding sites, and a C-terminal variable region that contributes to the differences among there isoforms.

The CH domain was found in a number of actin-binding proteins (such as α-actinin, spectrin, and filamin) to form the actin-binding region or serve as a regulatory structure.^[9] However, the CH domain in calponin is not the binding site for actin nor does it regulate the modes of calponin-F-actin binding.^[10] Nonetheless, CH domain in calponin was found to bind to extra-cellular regulated kinase (ERK) for calponin to play a possible role as an adaptor protein in the ERK signaling cascades.^[11]

Calponin binds actin to promote and sustain polymerization. The binding of calponin to F-actin inhibits the MgATPase activity of smooth muscle myosin.^{[12][13][12][14]} Calponin binds F-actin through two sites at residues 144-162 and 171-188 in chicken calponin 1. The two actin-binding sites are conserved in the three calponin isoforms.

There are three repeating sequence motifs in calponin next to the C-terminal region. This repeating structure is conserved in all three isoforms and across species. Outlined in Fig. 2, the first repeating motif overlaps with the second actin-binding site and contains protein kinase C (PKC) phosphorylation sites Ser175 and Thr184 that are not present in the first actin-binding site. This feature is consistent with the hypothesis that the second actin-binding site plays a regulatory role in the binding of calponin to the actin filament. Similar sequences as well as potential phosphorylation sites are present in repeats 2 and 3 whereas their function is unknown.

The C-terminal segment of calponin has diverged significantly among the three isoforms. The variable lengths and amino acid sequences of the C-terminal segment produce the size and charge differences among the calponin isoforms. The corresponding charge features rendered calponin 1, 2 and 3 the names of basic, neutral and acidic calponins.^[15][16][17]

The C-terminal segment of calponin has an effect on weakening the binding of calponin to F-actin. Deletion of the C-terminal tail strongly enhanced the actin-binding and bundling activities of all three isoforms of calponin.^[18][19] The C-terminal tail regulates the interaction with F-actin by altering the function of the second actin-bing site of calponin.^[20]

Numerous in vitro experimental data indicate that calponin 1 functions as an inhibitory regulator of smooth muscle contractility through inhibiting actomyosin interactions.^[2][21][22] In this regulation, binding of Ca²⁺-calmodulin and PKC phosphorylation dissociate calponin 1 from the actin filament and facilitate smooth muscle contraction.^[23]

In vivo data also support the role of calponin 1 as regulator of smooth muscle contractility. While aortic smooth muscle of adult Wistar Kyoto rats, which naturally lacks calponin 1, is fully contractile, it has a decreased sensitivity to norepinephrine activation.^[24][25] Matrix metalloproteinase-2 proteolysis of calponin 1 resulted in vascular hypocontractility to phenylephrine.^[26] Vas deferens smooth muscle from calponin 1 knockout mice showed faster maximum shortening velocity.^[27] Calponin 1 knockout mice exhibited blunted MAP response to phenylephrine administration.^[28]

There is a large collection of in vitro evidences demonstrating the phosphorylation regulation of calponin. The primary phosphorylation sites are Ser175 and Thr184 in the second actin-binding site (Fig. 2). Experimental data showed that Ser175 and Thr184 in calponin 1 are phosphorylated by PKC in vitro.^[23] Direct association was found between calponin 1 and PKCα^[29] and PKCε.^[11] Calmodulin-dependent kinase II and Rho-kinase are also found to phosphorylate calponin at Ser175 and Thr184 in vitro.^[30][31] Of these two residues, the main site of regulatory phosphorylation by calmodulin-dependent kinase II and Rho-kinase is Ser175. Dephosphorylation of calponin is catalyzed by type 2B protein phosphatase^[32][33]

Unphosphorylated calponin binds to actin and inhibits actomyosin MgATPase. Ser175 phosphorylation alters the molecular conformation of calponin and dissociates calponin from F-actin.^[34] The consequence is to release the inhibition of actomyosin MgATPase and increase the production of force.^[14][35][36]

Despite the overwhelming evidence for the phosphorylation regulation of calponin obtained from in vitro studies, phosphorylated calponin is not readily detectable in vivo or in living cells under physiological conditions.^[37][38] Based on the observation that PKC phosphorylation of calponin 1 weakens the binding affinity for the actin filaments,^[34] the phosphorylated calponin may not be stable in the actin cytoskeleton thus be degraded in the cell.