The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death.

The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.

S-palmitoylation and s-oleoylation of rabbit and pig sarcolipin.

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.

Nicotinic acetylcholine receptors in health and disease.

Nicotinic acetylcholine receptors (AChRs) are a family of acetylcholine-gated cation channels that form the predominant excitatory neurotransmitter receptors on muscles and nerves in the peripheral nervous system. AChRs are also expressed on neurons in lower amounts throughout the central nervous system. AChRs are even being reported on unexpected cell types such as keratinocytes. Structures of these AChRs are being determined with increasing precision, but functions of some orphan subunits are just beginning to be established. Functional roles for postsynaptic AChRs in muscle are well known, but in neurons the post-, peri-, extra-, and presynaptic roles of AChRs are just being revealed. Pathogenic roles of AChRs are being discovered in many diseases involving mechanisms ranging from mutations, to autoimmune responses, to the unknown; involving cell types ranging from muscles, to neurons, to keratinocytes; and involving signs and symptoms ranging from muscle weakness to epilepsy, to neurodegenerative disease, to psychiatric disease, to nicotine addiction. Awareness of AChR involvement in some of these diseases has provoked new interests in development of therapeutic agonists for specific AChR subtypes and the use of expressed cloned AChR subunits as possible immunotherapeutic agents. Highlights of recent developments in these areas will be briefly reviewed.

Epsilon subunit-containing acetylcholine receptors in myotubes belong to the slowly degrading population.

Two types of muscle acetylcholine receptors (AChRs) can be distinguished on the basis of their degradation rates and sensitivities to innervation, muscle activity, and agents elevating intracellular cAMP. The first type (Rs), is present in a stable form (degradation t1/2 = approximately 10 d) at the adult innervated neuromuscular junctions (NMJs). Rs can also exist in a less stable form (called accelerated Rs; t1/2 = approximately 3-5 d) at denervated NMJs and in aneurally cultured myotubes; agents that increase intracellular cAMP reversibly modulate Rs stability. The second type of AChR is a rapidly degrading receptor (Rr) expressed only in embryonic and noninnervated muscles. Rr can be stabilized by ATP and not by cAMP. This study tested the hypothesis that the degradation properties unique to the Rs are attributable to the presence of the epsilon subunit. Immunoprecipitation and Western blot analysis of AChRs extracted from rat muscle cells in tissue culture showed that AChRs recognized by antibodies against the epsilon subunit degraded as a single population with a half-life similar to that of the slow component, Rs, in these cells. In addition, as for Rs receptors in denervated NMJs and cultured muscle cell, the degradation rate of these epsilon-containing AChRs was stabilized by dibutyryl-cAMP. The data indicate that the epsilon-containing AChRs behave like Rs. Thus, the presence of the epsilon subunit is sufficient for selecting an AChR molecule to the Rs pool.

Differential titin isoform expression in human skeletal muscle.

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW approximately 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (mean +/- SE; age = 25.4 +/- 2.9 years, height = 177.7 +/- 1.8 cm, weight = 76.5 +/- 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n = 10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n = 5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P > 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.

Molecular diversity and functional characterization of voltage-dependent calcium channels (CACN4) expressed in pancreatic beta-cells.

Dihydropyridine-sensitive voltage-dependent calcium channels (VDCC) play a crucial role in insulin secretion. We recently have cloned a human alpha 1-subunit of the VDCC expressed in pancreatic beta-cells, designated CACN4. In this study we have isolated complementary DNAs encoding two forms of rat CACN4 (rCACN4A and rCACN4B) from a rat insulinoma RINm5F complementary DNA library. Rat CACN4A is a protein of 2203 amino acids and is the rat homolog of human CACN4, whereas rCACN4B lacks 535 amino acids in the carboxyl-terminal region, probably due to alternative splicing. We have found two additional variations, one in the intracellular loop between repeats I and II and the other in the extracellular region between the third and fourth segments of repeat IV. Reverse transcriptase-polymerase chain reaction analysis of rat pancreatic islet messenger RNA reveals that these variants are present in pancreatic islets. In addition, whole-cell voltage-clamp recordings of Chinese hamster ovary cells stably expressing the alpha 1-subunit (rCACN4A or rCACN4B) with or without the calcium channel beta 2-subunit show that coexpression of rCACN4A with the beta 2-subunit or rCACN4B with the beta 2-subunit elicits L-type VDCC currents, whereas expression of the alpha 1-subunit alone does not, indicating that CACN4 can associate functionally with the beta 2-subunit and that the beta-subunit is essential for functional expression of CACN4. These results suggest that there are various subtypes of CACN4 expressed in pancreatic beta-cells, and that both rCACN4A and rCACN4B can function as VDCC. Furthermore, the present study suggests that the expression of the beta-subunit as well as the alpha 1-subunit may participate in the regulation of insulin secretion.

Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains.

On the basis of similarities in sequence and structure, the protein domains that form the immunoglobulin superfamily have been divided into three sets: one with variable-like domains, the V set, and two with different variants of the constant-like domains, the C1 and C2 sets. Examination of a muscle member of the immunoglobulin superfamily, telokin, shows that its structure is closely related to those of the variable domains found in antibodies, CD2, CD4 and CD8. However, it also contains structural features that, previously, have only been found in constant domains. Telokin represents a new structural set in the superfamily which we call the I set. Using the structures of telokin, and variable domains from antibodies, CD4 and CD8, we constructed a profile that describes the sequence characteristics of the structural core common to those proteins. This sequence profile makes a good match to the sequences of many of the immunoglobulin superfamily domains that form the cell adhesion molecules and surface receptors. This match implies that these domains also have structures that belong to the I set.

Myofibrillar protein from different muscle fiber types: implications of biochemical and functional properties in meat processing.

Texture, moisture retention, and tenderness of processed muscle foods are influenced by the functionality of myofibrillar protein. Recent studies have revealed large variations in processing quality between red and white muscle groups that can be attributed to differences in the functional properties of myofibrillar protein associated with the type of fiber. Myofibrillar proteins from fast- and slow-twitch fibers exhibit different biochemical and rheological characteristics and form gels with distinctly different viscoelastic properties and microstructures. The existence and wide distribution of the numerous myosin isoforms in different muscle and fiber types contribute to the various functional behaviors of myofibrillar protein. The different sensitivities of fast and slow myofibrillar proteins to pH, ionic environment, temperature, and other external factors have been well documented and illustrate the importance of adjusting meat processing conditions, according to fiber type profile to achieve maximum protein functionalities, and hence, uniform quality of the final muscle foods.

Role of ryanodine receptors.

Recent findings on the ryanodine receptor of vertebrates, a Ca-release channel protein for the caffeine- and ryanodine-sensitive Ca pools, are reviewed in this article. Three distinct genes, i.e., ryr1, ryr2, and ryr3, express different isoforms in specific locations: Ryr1 in skeletal muscle and Purkinje cells of cerebellum; Ryr2 in cardiac muscle and brain, especially cerebellum; Ryr3 in skeletal muscle of nonmammalian vertebrates, the corpus striatum, and limbic cortex of brain, smooth muscles, and the other cells in vertebrates. While only one isoform (Ryr1) is expressed in mammalian skeletal muscles, two isoforms (alpha- and beta-isoforms expressed by ryr1 and ryr3, respectively) are found in nonmammalian vertebrate skeletal muscles. Although the coexistence of two isoforms may merely be related to differentiation and specialization, the biological significance remains to be clarified. Ryanodine receptors in vertebrate skeletal muscles are believed to mediate two different modes of Ca release: Ca(2+)-induced Ca release and action potential-induced Ca release. All results obtained so far with any isoform of ryanodine receptor are related to Ca(2+)-induced Ca release and show very similar characteristics. Ca(2+)-induced Ca release, however, cannot be the underlying mechanism of Ca release on skeletal muscle activation. Susceptibility of the ryanodine receptor's ryanodine-binding activity to modification by physical factors, such as osmolality of the medium, might be related to action potential-induced Ca release. A hypothesis of molecular interaction in view of the plunger model of action potential-induced Ca release is discussed, suggesting that the model could be compatible with Ryr1 and Ryr3, but incompatible with Ryr2. The functional relevance of ryanodine receptor isoforms, especially Ryr3, in brain also remains to be clarified. Among ryr1 gene-related diseases, malignant hyperthermia was the first to be identified; however, there is still the possibility of involvement of the other genes. Central core disease has been added to the list recently. A molecular approach for the diagnosis and treatment of diseases is now in progress.