Novel mutations at carboxyl terminus of CIC-1 channel in myotonia congenita.

OBJECTIVES: Myotonia congenita (MC), caused by mutations in the muscle chloride channel (CLCN1) gene, can be inherited dominantly or recessively. The mutations at the carboxyl terminus of the CLCN1 gene have been identified in MC patients, but the functional implication of these mutations is unknown. MATERIAL AND METHODS: Direct sequencing of polymerase chain reaction products covering the whole coding region of the CLCN1 gene was performed in a MC family. This study was designed to investigate the clinical manifestations and genetic analysis of the CLCN1 gene. RESULTS: We identified two novel mutations, 2330delG and 1892C>T, from a genetic screening of the CLCN1 gene in the MC family. The 2330delG mutant allele producing a fs793X truncated protein was identified in a heterozygous state in all the patients. The 1892C>T nucleotide change induced a missense mutation (T631I) found in several asymptomatic individuals, indicating that it may not be associated with MC. Intriguingly, the 2330delG mutation was also found in an asymptomatic subject who also carried the 1892C>T mutation. CONCLUSION: The data indicate that the fs793X mutant protein causes dominantly inherited MC. Because the mutation has been found in a recessive pedigree, the fs793X mutation may have a dual inheritance pattern.

Mutations at the arginine residues in alpha8 loop of Bacillus thuringiensis delta-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

The functional role of the alpha8 loop residues in domain II of Bacillus thuringiensis Cry1Ac toxin was examined. Alanine substitution mutations were introduced in the residues from 275 to 293. Among the mutant toxins, substitutions at R281 and R289 affected toxicity to Manduca sexta and Lymantria dispar. Loss of toxicity by these mutant toxins was well correlated with reductions in binding affinity for brush border membrane vesicles and the purified receptor, aminopeptidase N (APN), from both insects. These data suggest that the two arginine residues in the alpha8 loop region are important in toxicity and APN binding in L. dispar and M. sexta.

Identification of residues in domain III of Bacillus thuringiensis Cry1Ac toxin that affect binding and toxicity.

Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues (503)SS(504), (506)NNI(508), (509)QNR(511), (522)ST(523), and (524)ST(525). Single alanine substitutions were made at the residues (509)Q, (510)N, (511)R, and (513)Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations, (503)SS(504)-AA, (506)NNI(508)-AAA, (522)ST(523)-AA, (524)ST(525)-AA, and (510)N-A affected neither the protein's toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the (509)QNR(511)-AAA, (509)Q-A, (511)R-A, and (513)Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the (509)QNR(511)-AAA, (509)Q-A, (511)R-A, and (513)Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only (509)QNR(511)-AAA and (511)R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues (509)Q, (511)R, and (513)Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.

Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar.

Two amino acids, Gly and Ser, at positions 282 and 283 in the loop region of domain II of Cry1Ab2 toxin are substituted with Ala and Leu in the Cry1Ab9-033 toxin. Cry1Ab2 exhibited about a 10-fold increase in toxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua. Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDa L dispar BBMV protein. No measurable differences in dissociation binding assays were observed between these two toxins. Digestion of these toxins with L dispar gut enzymes and BBMV proteases indicated no differences in stability. Ala and Leu residues in Cry1Ab9-033 were substituted with Gly and Ser by site-directed mutagenesis to produce mutant Cry1Ab alpha 8. This toxin exhibited full recovery of toxicity and binding affinity for L dispar. These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity in L dispar.

Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.