Glycosylation of ß1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels.

BACKGROUND: The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. METHODS: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. RESULTS: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. CONCLUSION: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

Glycosylation of ß1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. Results: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.(AU)

Glycosylation of ß1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. Results: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.(AU)

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 μg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 μg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 μg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

Comparison of wavefront aberrations under cycloplegic, scotopic and photopic conditions using WaveScan.

PURPOSE: To evaluate the differences of wavefront aberrations under cycloplegic, scotopic and photopic conditions. METHODS: A total of 174 eyes of 105 patients were measured using the wavefront sensor (WaveScan® 3.62) under different pupil conditions: cycloplegic 8.58 ± 0.54 mm (6.4 mm - 9.5 mm), scotopic 7.53 ± 0.69 mm (5.7 mm - 9.1 mm) and photopic 6.08 ± 1.14 mm (4.1 mm - 8.8 mm). The pupil diameter, standard Zernike coefficients, root mean square of higher-order aberrations and dominant aberrations were compared between cycloplegic and scotopic conditions, and between scotopic and photopic conditions. RESULTS: The pupil diameter was 7.53 ± 0.69 mm under the scotopic condition, which reached the requirement of about 6.5 mm optical zone design in the wavefront-guided surgery and prevented measurement error due to the pupil centroid shift caused by mydriatics. Pharmacological pupil dilation induced increase of standard Zernike coefficients Z(3)(-3), Z(4)(0) and Z(5)(-5). The higher-order aberrations, third-order aberration, fourth-order aberration, fifth-order aberration, sixth-order aberration, and spherical aberration increased statistically significantly, compared to the scotopic condition (P<0.010). When the scotopic condition shifted to the photopic condition, the standard Zernike coefficients Z(4)(0), Z(4)(2), Z(6)(-4), Z(6)(-2), Z(6)(2) decreased and all the higher-order aberrations decreased statistically significantly (P<0.010), demonstrating that accommodative miosis can significantly improve vision under the photopic condition. Under the three conditions, the vertical coma aberration appears the most frequently within the dominant aberrations without significant effect by pupil size variance, and the proportion of spherical aberrations decreased with the decrease of the pupil size. CONCLUSIONS: The wavefront aberrations are significantly different under cycloplegic, scotopic and photopic conditions. Using the wavefront sensor (VISX WaveScan) to measure scotopic wavefront aberrations is feasible for the wavefront-guided refractive surgery.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy.

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 µg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 µg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 µg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

Comparison of wavefront aberrations under cycloplegic, scotopic and photopic conditions using WaveScan / Comparação de aberrações de frente de onda sob cicloplegia, condições escotópica e fotópica usando WaveScan

PURPOSE: To evaluate the differences of wavefront aberrations under cycloplegic, scotopic and photopic conditions. METHODS: A total of 174 eyes of 105 patients were measured using the wavefront sensor (WaveScan® 3.62) under different pupil conditions: cycloplegic 8.58 ± 0.54 mm (6.4 mm - 9.5 mm), scotopic 7.53 ± 0.69 mm (5.7 mm - 9.1 mm) and photopic 6.08 ± 1.14 mm (4.1 mm - 8.8 mm). The pupil diameter, standard Zernike coefficients, root mean square of higher-order aberrations and dominant aberrations were compared between cycloplegic and scotopic conditions, and between scotopic and photopic conditions. RESULTS: The pupil diameter was 7.53 ± 0.69 mm under the scotopic condition, which reached the requirement of about 6.5 mm optical zone design in the wavefront-guided surgery and prevented measurement error due to the pupil centroid shift caused by mydriatics. Pharmacological pupil dilation induced increase of standard Zernike coefficients Z3-3, Z4(0) and Z5-5. The higher-order aberrations, third-order aberration, fourth-order aberration, fifth-order aberration, sixth-order aberration, and spherical aberration increased statistically significantly, compared to the scotopic condition (P<0.010). When the scotopic condition shifted to the photopic condition, the standard Zernike coefficients Z4(0), Z4², Z6-4, Z6-2, Z6² decreased and all the higher-order aberrations decreased statistically significantly (P<0.010), demonstrating that accommodative miosis can significantly improve vision under the photopic condition. Under the three conditions, the vertical coma aberration appears the most frequently within the dominant aberrations without significant effect by pupil size variance, and the proportion of spherical aberrations decreased with the decrease of the pupil size. CONCLUSIONS: The wavefront aberrations are significantly different under cycloplegic, scotopic and photopic conditions. Using the wavefront sensor (VISX WaveScan) to measure scotopic wavefront aberrations is feasible for the wavefront-guided refractive surgery.

OBJETIVO: Avaliar as diferenças de aberrações de frente de onda, em diferentes condições pupilares: sob cicloplegia, escotópica e fotópica. MÉTODOS: Um total de 174 olhos de 105 pacientes foram avaliados utilizando o sensor de frente de onda (WaveScan® 3.62) em diferentes condições pupilares: sob cicloplegia 8,58 ± 0,54 mm (6.4 mm-9.5 mm), escotópica 7,53 ± 0,69 mm (5,7 mm - 9,1 mm) e fotópica 6,08 ± 1,14 mm (4,1 mm - 8,8 mm). Diâmetro da pupila, coeficientes de Zernike, RMS ("Root Mean Square") das aberrações de alta ordem e as aberrações dominantes foram comparados entre as condições sob cicloplegia e escotópica, e entre as condições escotópica e fotópica. RESULTADOS: O diâmetro da pupila foi 7,53 ± 0.69 mm sob a condição escotópica e atingiu a exigência de cerca de 6,5 mm de zona óptica na cirurgia baseada em análise de frentes de ondas, evitando erros de medição consequentes à mudança de centroide pupilar provocada por midriáticos. A dilatação farmacológica da pupila induziu aumento dos coeficientes de Zernike Z3-3, Z4(0) e Z5-5. As aberrações de mais alta ordem (terceira, quarta, quinta e sexta ordem) e a aberração esférica aumentaram de forma estatisticamente significativa, em comparação com a condição escotópica (P<0,010). Quando a condição escotópica se mudou para a condição fotópica, os coeficientes de Zernike Z4(0), Z4², Z6-4, Z6-2, Z6² e todas as aberrações de alta ordem diminuíram de forma estatisticamente significativa (P<0,010), demonstrando que a miose acomodativa pode significativamente melhorar a visão sob a condição fotópica. Sob as três condições, a aberração coma vertical apareceu mais frequentemente dentro das aberrações dominantes, sem influência significativa da variação do tamanho da pupila, e a proporção de aberração esférica diminuiu com a diminuição do tamanho da pupila. CONCLUSÕES: As aberrações de frente de onda são significativamente diferentes sob cicloplegia, condições escotópica e fotópica. O uso do sensor de frentes de onda (VISX WaveScan) para medir as aberrações de frente de onda escotópicas é uma opção viável para a cirurgia refrativa baseada em análise de frentes de onda.