The pathomechanism of familial hypokalemic periodic paralysis (HypoPP) is a mystery, despite knowledge of the underlying dominant point mutations in the dihydropyridine receptor (DHPR) voltage sensor. In five HypoPP families without DHPR gene defects, we identified two mutations, Arg-672→His and →Gly, in the voltage sensor of domain 2 of a different protein: the skeletal muscle sodium channel α subunit, known to be responsible for hereditary muscle diseases associated with myotonia. Excised skeletal muscle fibers from a patient heterozygous for Arg-672→Gly displayed depolarization and weakness in low-potassium extracellular solution. Slowing and smaller size of action potentials were suggestive of excitability of the wild-type channel population only. Heterologous expression of the two sodium channel mutations revealed a 10-mV left shift of the steady-state fast inactivation curve enhancing inactivation and a sodium current density that was reduced even at potentials at which inactivation was removed. Decreased current and small action potentials suggested a low channel protein density. The alterations are decisive for the pathogenesis of episodic muscle weakness by reducing the number of excitable sodium channels particularly at sustained membrane depolarization. The results prove that SCN4A, the gene encoding the sodium channel α subunit of skeletal muscle is responsible for HypoPP-2 which does not differ clinically from DHPR-HypoPP. HypoPP-2 represents a disease caused by enhanced channel inactivation and current reduction showing no myotonia.

Hypokalaemic periodic paralysis (hypoPP) is a dominantly inherited muscle disorder characterized by episodes of flaccid weakness. Previous genetic studies revealed mutations in the voltage-gated calcium channel alpha1-subunit (CACNA1S gene) in families with hypoPP (type I). Electrophysiological studies on these mutants in different expression systems could not explain the pathophysiology of the disease. In addition, several mutations (Arg669His, Arg672His, Arg672Gly and Arg672Ser) in the voltage sensor of the skeletal muscle sodium channel alpha-subunit (SCN4A gene) have been found in families with hypoPP (type II). For Arg672Gly/His a fast inactivation defect was described, and for Arg669His an impairment of slow inactivation was reported. Except for the substitution for serine, we have now expressed all mutants in a human cell-line and studied them electrophysiologically. Patch-clamp recordings show an enhanced fast inactivation for all three mutations, whereas two of them reveal enhanced slow inactivation. This may reduce the number of functional sodium channels at resting membrane potential and contribute to the long-lasting periods of paralysis experienced by hypoPP patients. The gating of both histidine mutants (Arg669His, Arg672His) can be modulated by changes of extra- or intracellular pH. The inactivation defects of Arg669His and Arg672His can be alleviated by low pH to a significant degree, suggesting that the decrease of pH in muscle cells (e.g. during muscle work) might lead to an auto-compensation of functional defects. This may explain a delay or prevention of paralytic attacks in patients by slight physical activity. Moreover, the histidine residues may be the target for a potential therapeutic action by acetazolamide.

Hypokalemic periodic paralysis type 1 (HypoPP-1) is a hereditary muscular disorder caused by point mutations in the gene encoding the voltage-gated Ca(2+) channel alpha subunit (Ca(v)1.1). Despite extensive research, the results on HypoPP-1 mutations are minor and controversial, as it is difficult to analyse Ca(2+) channel activation macroscopically due to an existence of two open states. In this study, we heterologously expressed the wild-type and HypoPP-1 mutations introduced into the rabbit cardiac Ca(2+) channel (R650H, R1362H, R1362G) in HEK-293 cells. To examine the cooperative effects of the mutations on channel gating, we expressed two double mutants (R650H/R1362H, R650H/R1362G). We performed whole-cell patch-clamp and, to obtain more information, applied a global fitting procedure whereby several current traces elicited by different potentials were simultaneously fit to the kinetic model containing four closed, two open and two inactivated states. We found that all HypoPP-1 mutations have "loss-of-function" features: D4/S4 mutations shift the equilibrium to the closed states, which results in reduced open probability, shorter openings and, therefore, in smaller currents, and the D2/S4 mutant slows the activation. In addition, HypoPP-1 histidine mutants favored the second open state O(2) with a possibly lower channel selectivity. Cooperativity between the D2/S4 and D4/S4 HypoPP-1 mutations manifested in dominant effects of the D4/S4 mutations on kinetics of the double mutants, suggesting different roles of D2/S4 and D4/S4 voltage sensors in the gating of voltage-gated calcium channels.

BackgroundTo explore the relationship between the heart-type fatty acid binding protein (H-FABP) gene and intramuscular fat (IMF), a polymorphism of the second exon of the H-FABP gene was investigated in 60 Three-yellow chickens (TYCs) and 60 Hetian-black chickens (HTBCs).ResultsThe IMF contents of the cardiac, chest and leg muscles in HTBC were increased compared with TYC. Both TYC and HTBC populations exhibited Hardy-Weinberg Equilibrium (HWE) according to the χ2 test. Three variations of the two birds were detected, namely, G939A, G982A and C1014T. HTBCs with the TT genotypes exhibit increased IMF content in the chest muscles compared with the TC genotype. Thus, the G982A site could be considered a genetic marker for selecting increased IMF content in the chest muscles of HTBC. The correlation coefficients revealed that H-FABP mRNA expression was negatively correlated with the IMF content in the cardiac, chest and leg muscles of HTBC and in the cardiac and chest muscles of TYC. The relative mRNA expression of H-FABP was reduced in the cardiac and leg muscles of HTBC compared with TYC, but this difference was not observed at the protein level, as assessed by Western blot analysis.ConclusionsThese findings offer essential data that can be useful in the breeding program of HTBC and future research exploring the role of H-FABP in IMF deposition and regulation in chickens.

L-type calcium-channel mutations causing hypokalemic periodic paralysis type 1 (HypoPP-1) have pronounced "loss-of-function" features and stabilize the less-selective second open state O(2), as we demonstrated in the companion paper. Here, we compared the effects of the L-type calcium-channel activator (+/-)BayK 8644 (BayK) on the heterologously expressed wild-type (WT) calcium channel, rabbit Cav1.2 HypoPP-1 analogs, and two double mutants (R650H/R1362H, R650H/R1362G). Our goal was to elucidate (1) whether the "loss-of-function" in HypoPP-1 can be compensated by BayK application, (2) how the less-selective open state is affected by BayK in WT and HypoPP-1 mutants, as well as (3) to gain an insight into BayK mechanism of action. Ionic currents were examined by whole-cell patch-clamp and analyzed by the global-fitting procedure. Our results imply that (1) BayK promotes channel activation, but equalized the differences among the WT and mutants, thus attenuating HypoPP-related effects on activation and deactivation; (2) BayK binds to the first open state O(1), and then serves as a catalyst for O(2) formation; (3) binding of BayK is impaired in the HypoPP mutants, thus affecting the formation of the less-selective second open state; (4) BayK affects cooperativity between the single HypoPP-1 mutations at all stages of the channel gating; and (5) BayK favoring of O(2) lowers calcium-channel selectivity.

This study aims to assess the association of polymorphisms and mRNA expression of adipocyte‐type fatty acid‐binding protein (A‐FABP) with intramuscular fat (IMF) in the breast muscle (BM) and leg muscle (LM) of Baicheng‐You chickens (BYCs). A total of 180 chickens, including sixty black Baicheng‐You chickens (BBYCs), sixty silky Baicheng‐You chickens (SBYCs) and sixty white Baicheng‐You chickens (WBYCs), were reared from 1 to 120 day. A polymerase chain reaction–single‐strand conformation polymorphism strategy (PCR‐SSCP) was used to detect the polymorphism of the A‐FABP gene in the first exon, and the C51T silent mutational site was found. The IMF content with the AA genotype was significantly higher than that with the AG genotype (p = 0.0473) in the LM of WBYC. Thus, this site could be taken as a molecular marker in selecting a higher IMF content of LM in WBYC. A‐FABP gene mRNA expression in the BM and LM of BYCs was detected, and a significant positive correlation was observed in the LM of WBYC. These findings provide fundamental data that might be useful in further study of the role of the A‐FABP gene in IMF content and fatty metabolism in chickens.

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Lipoprotein lipase (LPL) was often taken as a candidate gene for investigating fat metabolism. However, there are few studies on the effect of LPL on intramuscular fat (IMF) deposition in Baicheng oil chicken (BOC) and Three‐yellow Chicken (TYC). In this study, we studied the relationship between polymorphism and messenger RNA (mRNA) expression of LPL with IMF deposition in the chest muscle (CM) and leg muscle (LM) of TYC and BOC. Sixty TYCs and 60 BOCs were raised from 1 d and slaughtered by avascularization at their slaughtering age. IMF contents of the CM and LM in the BOC were markedly higher than those in the TYC. Three genotypes following AA, AB and BB were found by the method of polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP). The synonymous mutation C12315T was detected. The content of IMF with the AA genotype was significantly higher than the AB genotype in the LM of TYC. The mRNA expression both of CM and LM in BOC was prominently higher than those in TYC, and there was a positive significant correlation between LM and CM in both BOC and TYC. These results suggested that the SNPs polymorphism and mRNA expression of the LPL gene might be helpful for selective breeding in IMF of the chicken.