Kv channel interacting proteins (KChIPs) are Ca2+-binding proteins with four EF-hands and well-known to modulate Kv4.2 channel gating. The present study is carried out to investigate the molecular mechanism related to regulate the interaction of KChIP2.2 and KChIP4a with Kv channel. In comparison with KChIP4a, the interaction of KChIP2.2 with Kv4.2 was more obvious in the absence of Ca2+ or Mg2+. However the binding of KChIP2.2 and KChIP4a toward Kv4.2 increased with increasing Ca2+ and Mg2+ concentration. Nevertheless, no individual regions within KChIP2.2 and KChIP4a could exclusively fulfill the interaction between KChIPs mutants and Kv channel. Fluorescence measurement showed that KChIP2.2 possessed both high affinity and low affinity Ca2+-binding sites, but only low affinity Ca2+-binding site was observed with KChIP4a. However, both of them have only one Mg2+-binding site. Studies on the truncated mutants revealed that the EF-hand 4 of KChIP2.2 was related to high affinity binding with Ca2+, and the integrity of molecular structure of KChIP2.2 and KChIP4a was important for Ca2+ -and Mg2+-binding. The thermal stability of KChIP2.2 and KChIP4 was found to be differentiately affected by Ca2+ and Mg2+. Proteolytic digestion and thiol reactivity assays also supported that Ca2+ and Mg2+-induced conformational change of KChIP2.2 was differed from KChIP4a. Moreover, in cells co-transfected with Kv4.2 cDNA, it was formed that KChIP2.2 trafficking to the cell surface was increased by elevating intracellular Ca2+ concentration, but no noticeable change was observed for KChIP4a. Taken together, these results suggest that the conformational changes of KChIP2.2 and KChIP4a differently induced by Ca2+ and Mg2+ affect their binding with Kv channel and/or cellular distribution.

In the nervous system, the roles of Kv channels are well established as being critical for regulating action potential frequency, membrane potential, and neurotransmitter release. However, their role in glial cells, a non-excitable cell type, is yet to be fully understood. Whole-cell current kinetics, pharmacology, immunocytochemistry and RT-PCR were used to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ~70%, 10% and less than 5% of total A current is associated with Kv4, Kv3 and Kv1 channels, respectively. In addition, pharmacology and kinetics provide novel evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Since astrocytes are known to be subjected to neuronal firing frequencies of up to 200 Hz in the hippocampus, the role of A currents in membrane voltage oscillations was assessed. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. Astrocytes and HEK293 cells were used to investigate the mechanism of the previously found GABAA induced anion-mediated reduction of Kv channels in more detail. Astrocytes demonstrate an anion concentration specific depolarizing effect on inactivating A-type (also termed transient voltage-gated) K+ channel activation kinetics whereas a hyperpolarizing effect was seen upon expression of Kv4.2 or Kv1.4 in HEK293 cells, but only after disruption of the cytoskeleton using cytochalasin D. It is hypothesized that cytoskeletal interactions and Cl -mediated effects are mediated through N-terminal conformational stabilities. <p>In summary, the results indicate that hippocampal astrocytes in vitro express multiple A type Kv channel á subunits with accessory, Ca2+-sensitive cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Functions of these channels remain to be determined in a physiological setting, but suggest that A-type Kv channels enable astrocytes to respond rapidly with membrane voltage oscillations to high frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. Furthermore, studies of anion and cytoskeletal effects on Kv channels demonstrate channel function to be highly localized/targeted and susceptible to changes in ionic environment.

In the nervous system, the roles of Kv channels are well established as being critical for regulating action potential frequency, membrane potential, and neurotransmitter release. However, their role in glial cells, a non-excitable cell type, is yet to be fully understood. Whole-cell current kinetics, pharmacology, immunocytochemistry and RT-PCR were used to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ~70%, 10% and less than 5% of total A current is associated with Kv4, Kv3 and Kv1 channels, respectively. In addition, pharmacology and kinetics provide novel evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Since astrocytes are known to be subjected to neuronal firing frequencies of up to 200 Hz in the hippocampus, the role of A currents in membrane voltage oscillations was assessed. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. Astrocytes and HEK293 cells were used to investigate the mechanism of the previously found GABAA induced anion-mediated reduction of Kv channels in more detail. Astrocytes demonstrate an anion concentration specific depolarizing effect on inactivating A-type (also termed transient voltage-gated) K+ channel activation kinetics whereas a hyperpolarizing effect was seen upon expression of Kv4.2 or Kv1.4 in HEK293 cells, but only after disruption of the cytoskeleton using cytochalasin D. It is hypothesized that cytoskeletal interactions and Cl -mediated effects are mediated through N-terminal conformational stabilities. <p>In summary, the results indicate that hippocampal astrocytes in vitro express multiple A type Kv channel á subunits with accessory, Ca2+-sensitive cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Functions of these channels remain to be determined in a physiological setting, but suggest that A-type Kv channels enable astrocytes to respond rapidly with membrane voltage oscillations to high frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. Furthermore, studies of anion and cytoskeletal effects on Kv channels demonstrate channel function to be highly localized/targeted and susceptible to changes in ionic environment.

Mutagenesis studies on the four EF-hands of KChIP2.2 (Potassium channel-interacting protein 2.2) were carried out to explore the conformational transition upon the binding of Ca2+ and Mg2+ and the subsequent effect on the interaction between KChIP2.2 and Kv4.2. CD spectra indicated that Ca2+- and Mg2+-loaded wild-type and mutated KChIP2.2 altered the secondary structure contents. In contrast to other mutants, mutation on EF1 caused a notably change in the secondary structure of KChIP2.2. Fluorescence measurement revealed that EF-hands 3 and 4 were high affinity Ca2+-binding sites within KChIP2.2 molecule, but the binding of Mg2+ with KChIP2.2 was marginally affected by EF-hand mutations. The results of size-exclusion chromatography showed that mutations on EF-1, EF-2 and EF-3 induced the oligomerization of KChIP2.2 and the extent of oligomerization was enhanced by Ca2+ and Mg2+. No significant differences were noted when wild-type and mutated KChIP2.2 bound with porcine brain membrane and liposome either in the absence or presence of Ca2+- and Mg2+. Pull down assay showed that KChIP2.2 and EF-hand mutants could bind with Kv4.2 in the absence of Ca2+ and Mg2+, but the interaction was enhanced by Ca2+ and Mg2+. However, the binding capability of mutants for Kv4.2 was notably lower than that observed for wild-type KChIP2.2. It was found that, in sharp contrast to that EF1 mutant exclusively localized in the nucleus, the other EF-hand mutants and wild-type protein distributed within nucleus as well as cytoplasm. Elevating intracellular Ca2+ concentration caused the translocation of EF1 mutant to cytoplasm but no appreciable effect on other mutants and wild-type KChIP2.2. . Taken together, these results suggest that the integrity of the four EF-hands are involved in function to stabilize conformation for binding with Kv channel, but this conformational transition is not essential for the binding to cell membrane.

Heterologous expression systems such as COS-7 cells have demonstrated the profound effects of KCNAB1-3 or Kvβ1-3 proteins on voltage gated potassium channels (Kv) channels. Indeed, in the presence of these β-subunits transiently expressed Kv channels are often modulated in multiple ways. Kv channel membrane expression is often increased in the presence of β-subunits. In addition, non-inactivating Kv currents suddenly become fast-inactivating and fast-inactivating channels become even faster. While much research has demonstrated the profound effects the β-subunits in particular the Kvβ1 subunit have on transiently expressed Kv currents little to date is known of the physiological role it may play. One study demonstrated that by “knocking out” Kvβ1 cardiomyocyte current changes were noted including a decrease in the Ito,f current. While this novel finding demonstrated a key cardiac physiological role of the Kvβ1 subunit it left many unanswered questions as to determine the cardiovascular regulation the Kvβ1 subunit provides. Indeed, cardiac arrhythmias and other electrical abnormalities within the heart such as long QT present patients with many unfortunate unknowns. Many of these incidences occur often abruptly with cardiac electrical abnormalities. Genetic research has begun to shine light on key cardiovascular genes in particular those coding for ion channels and auxiliary subunits or β-subunits. Kv channels and their β-subunits have gained particular notoriety in their key responsibility in restoring the resting membrane potential known as the repolarization phase. Indeed genetic manipulation and physiological examination of Kv channels and recently their β-subunits has demonstrated profound physiological results including prolonged QT durations within mice altered functional activity during physiological cycles such as estrus. While initial findings of Kvβ1 have demonstrated profound cellular and cardiomyocyte current alterations much still remains unknown. Therefore, this work hypothesizes that the Kvβ1 subunit provides a profound cardiovascular role in regulation and redox sensing at the physiological and pathophysiological level in both males and females. This work identifies a sex-based difference in cardiovascular regulation by Kvβ1 as well as demonstrated a profound redox sensing ability during altered metabolic states seen in pathophysiological conditions.

INTRODUÇÃO: O bócio é um termo genérico usado para descrever o aumento no volume da glândula tireoide que pode estar associado à formação de múltiplos nódulos, o chamado bócio multinodular. Camundongos transgênicos tireoide específicos com depleção do Pten ou aumento de expressão do Dream, dois importantes genes que têm sido implicados nas vias de sinalização das células foliculares, apresentam desenvolvimento de bócio. Em humanos, uma larga porcentagem de pacientes com doença de Cowden apresentam bócio ou outras anormalidades tireoidianas associadas a mutações germinativas no PTEN. OBJETIVO: O objetivo desse estudo foi investigar a expressão dos genes PTEN e DREAM em tecido hiperplásico tireoidiano, bem como mutações germinativas e somáticas no PTEN e mutações somáticas no DREAM, em pacientes portadores de bócio multinodular, com a finalidade de avaliar o papel destes genes na etiologia do bócio. MÉTODOS: Foram investigados 60 pacientes com bócio multinodular (54 mulheres). A extração do DNA genômico foi realizada a partir de tecido hiperplásico da tireoide e do sangue periférico dos pacientes enquanto o RNA foi obtido apenas do tecido glandular. A quantificação relativa do RNA mensageiro do PTEN e do DREAM foi avaliada pelo método de 2-??Ct utilizando o GAPDH como normalizador em dados produzidos pela PCR em tempo real. A alta e a baixa expressão de PTEN e DREAM foram definidas, respectivamente, por valores de quantificação superiores a 2.0 e inferiores a 0.5 em comparação a um pool comercial de RNA de tireoide normal de humanos. Análise de mutação foi realizada por amplificação da região codificante dos genes PTEN e DREAM pela PCR convencional seguida por sequenciamento automático (RQ = quantificação relativa; x? = média e DP = desvio padrão). RESULTADOS: Foi observada alta expressão do PTEN em 58,3% dos pacientes portadores de bócio (x RQ = 3,81; DP = 2,26) enquanto apenas dois casos apresentaram baixa expressão (x? RQ = 0,34; DP= 0,09). Nos 38,3% casos restantes foi observada expressão normal de PTEN (x? RQ = 1,35; DP = 0,35). Em relação ao gene DREAM, alta e baixa expressão foram observadas em 33,3% (x RQ = 6,07; DP = 5,02) e 15,0% (x RQ = 0,30; DP = 0,10) dos pacientes com bócio respectivamente, enquanto pouco mais da metade dos casos (51,6%) teve expressão normal RQ = 1,12 ; DP = 0,40). A Análise de mutações do PTEN e do DREAM revelaram apenas polimorfismos intrônicos, previamente descritos no banco de dados do NCBI, tanto nos DNA de sangue e/ou de tecido hiperplásico. CONCLUSÕES: Nossos resultados demonstraram uma expressão aumentada de PTEN em bócio multinodular, sugerindo que este pode estar hiperexpresso, ou pelo menos tem sua expressão mantida, nesta hiperplasia benigna da tireoide. Alterações na sequência gênica codificante do PTEN não foram observadas. Na análise mutacional e de expressão do DREAM não foram encontradas alterações que pudessem ser relacionadas à patogênese de bócio em humanos / BACKGROUND: Multinodular goiter is a clinicopathological entity characterized by an increased volume of the thyroid gland with formation of nodules. A high proliferative status of thyroid follicular cells and goiter were observed in mutants mice with specifically deleted Pten or Dream overexpression in thyrocytes. In humans, a large percentage of patients with Cowden disease have goiters or other thyroid abnormalities associated with germ-line PTEN mutations. OBJECTIVE: The aim of this study was to investigate the tissue expression of PTEN and DREAM, as well as germ-line and somatic PTEN mutations and somatic DREAM mutations, in patients with multinodular goiter to evaluated the role of these genes in goitrogenesis. METHODS: We investigated 60 multinodular goiter patients (54 females). Genomic DNA was extracted from both patients\' hyperplastic thyroid tissue and peripheral blood whereas RNA was obtained only from glandular tissue. Relative quantification of PTEN and DREAM messenger RNA was evaluated using 2-Ct method normalized to GAPDH expression on data produced by real-time PCR. PTEN and DREAM over and lower expression were respectively defined by value > 2.0-fold and < 0.5-fold relative to a commercial pool of normal human thyroid RNA. Mutations analyses were performed by amplification of PTEN and DREAM coding region by PCR followed by automatic sequencing. RQ = relative quantification; x = average; SD = standard deviation. RESULTS: We observed a high expression of PTEN in 58.3% of multinodular goiter patients (RQ x = 3.81; SD = 2.26) and only two cases with lower expression (RQ x = 0.34; SD = 0.09). In the remaining 38.3% of patients expression of PTEN was normal (RQ x = 1.35; SD = 0.35). For the DREAM, over and lower expression were observed in 33.3% (RQ x = 6.07; SD = 5.02) and 15.0% (RQ x = 0.30; SD = 0.10) of patients respectively, whereas 51.6% had normal expression (RQ x = 1.12; SD = 0.40). Regarding PTEN and DREAM mutations analysis, only previously described intronic polymorphisms were observed in DNA from blood and/or thyroid hyperplastic tissue. CONCLUSIONS: Our results demonstrated that PTEN expression is higher in multinodular goiter suggesting that this gene is overregulated (or at least has its expression maintained) in this benign hyperplastic thyroid lesions. No evidence for the involvement of DREAM in goitrogenesis was observed in our cohort of multinodular goiter patients

Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication

INTRODUÇÃO: O bócio é um termo genérico usado para descrever o aumento no volume da glândula tireoide que pode estar associado à formação de múltiplos nódulos, o chamado bócio multinodular. Camundongos transgênicos tireoide específicos com depleção do Pten ou aumento de expressão do Dream, dois importantes genes que têm sido implicados nas vias de sinalização das células foliculares, apresentam desenvolvimento de bócio. Em humanos, uma larga porcentagem de pacientes com doença de Cowden apresentam bócio ou outras anormalidades tireoidianas associadas a mutações germinativas no PTEN. OBJETIVO: O objetivo desse estudo foi investigar a expressão dos genes PTEN e DREAM em tecido hiperplásico tireoidiano, bem como mutações germinativas e somáticas no PTEN e mutações somáticas no DREAM, em pacientes portadores de bócio multinodular, com a finalidade de avaliar o papel destes genes na etiologia do bócio. MÉTODOS: Foram investigados 60 pacientes com bócio multinodular (54 mulheres). A extração do DNA genômico foi realizada a partir de tecido hiperplásico da tireoide e do sangue periférico dos pacientes enquanto o RNA foi obtido apenas do tecido glandular. A quantificação relativa do RNA mensageiro do PTEN e do DREAM foi avaliada pelo método de 2-??Ct utilizando o GAPDH como normalizador em dados produzidos pela PCR em tempo real. A alta e a baixa expressão de PTEN e DREAM foram definidas, respectivamente, por valores de quantificação superiores a 2.0 e inferiores a 0.5 em comparação a um pool comercial de RNA de tireoide normal de humanos. Análise de mutação foi realizada por amplificação da região codificante dos genes PTEN e DREAM pela PCR convencional seguida por sequenciamento automático (RQ = quantificação relativa; x? = média e DP = desvio padrão). RESULTADOS: Foi observada alta expressão do PTEN em 58,3% dos pacientes portadores de bócio (x RQ = 3,81; DP = 2,26) enquanto apenas dois casos apresentaram baixa expressão (x? RQ = 0,34; DP= 0,09). Nos 38,3% casos restantes foi observada expressão normal de PTEN (x? RQ = 1,35; DP = 0,35). Em relação ao gene DREAM, alta e baixa expressão foram observadas em 33,3% (x RQ = 6,07; DP = 5,02) e 15,0% (x RQ = 0,30; DP = 0,10) dos pacientes com bócio respectivamente, enquanto pouco mais da metade dos casos (51,6%) teve expressão normal RQ = 1,12 ; DP = 0,40). A Análise de mutações do PTEN e do DREAM revelaram apenas polimorfismos intrônicos, previamente descritos no banco de dados do NCBI, tanto nos DNA de sangue e/ou de tecido hiperplásico. CONCLUSÕES: Nossos resultados demonstraram uma expressão aumentada de PTEN em bócio multinodular, sugerindo que este pode estar hiperexpresso, ou pelo menos tem sua expressão mantida, nesta hiperplasia benigna da tireoide. Alterações na sequência gênica codificante do PTEN não foram observadas. Na análise mutacional e de expressão do DREAM não foram encontradas alterações que pudessem ser relacionadas à patogênese de bócio em humanos / BACKGROUND: Multinodular goiter is a clinicopathological entity characterized by an increased volume of the thyroid gland with formation of nodules. A high proliferative status of thyroid follicular cells and goiter were observed in mutants mice with specifically deleted Pten or Dream overexpression in thyrocytes. In humans, a large percentage of patients with Cowden disease have goiters or other thyroid abnormalities associated with germ-line PTEN mutations. OBJECTIVE: The aim of this study was to investigate the tissue expression of PTEN and DREAM, as well as germ-line and somatic PTEN mutations and somatic DREAM mutations, in patients with multinodular goiter to evaluated the role of these genes in goitrogenesis. METHODS: We investigated 60 multinodular goiter patients (54 females). Genomic DNA was extracted from both patients\' hyperplastic thyroid tissue and peripheral blood whereas RNA was obtained only from glandular tissue. Relative quantification of PTEN and DREAM messenger RNA was evaluated using 2-Ct method normalized to GAPDH expression on data produced by real-time PCR. PTEN and DREAM over and lower expression were respectively defined by value > 2.0-fold and < 0.5-fold relative to a commercial pool of normal human thyroid RNA. Mutations analyses were performed by amplification of PTEN and DREAM coding region by PCR followed by automatic sequencing. RQ = relative quantification; x = average; SD = standard deviation. RESULTS: We observed a high expression of PTEN in 58.3% of multinodular goiter patients (RQ x = 3.81; SD = 2.26) and only two cases with lower expression (RQ x = 0.34; SD = 0.09). In the remaining 38.3% of patients expression of PTEN was normal (RQ x = 1.35; SD = 0.35). For the DREAM, over and lower expression were observed in 33.3% (RQ x = 6.07; SD = 5.02) and 15.0% (RQ x = 0.30; SD = 0.10) of patients respectively, whereas 51.6% had normal expression (RQ x = 1.12; SD = 0.40). Regarding PTEN and DREAM mutations analysis, only previously described intronic polymorphisms were observed in DNA from blood and/or thyroid hyperplastic tissue. CONCLUSIONS: Our results demonstrated that PTEN expression is higher in multinodular goiter suggesting that this gene is overregulated (or at least has its expression maintained) in this benign hyperplastic thyroid lesions. No evidence for the involvement of DREAM in goitrogenesis was observed in our cohort of multinodular goiter patients