Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

이영미,Gareth A. Thompson,Ian Ashmole,Mark Leyland,Insuk So,Peter R. Stanfield 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba2+ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K+ currents in the absence and presence of externally applied Ba2+. Wild type Kir2.1 was blocked by externally applied Ba2+ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba2+ blockage. The estimated Kd(0) was 108μM for Kir2.1 wild type, 124μM for a concatameric WT-Y145V dimer, 109μM for a WT-Y145L dimer, and 267μM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba2+, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba2+ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba2+ blockage in Kir 2.1.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba<SUP>2+</SUP>

Young Mee Lee,Gareth A. Thompson,Ian Ashmole,Mark Leyland,Insuk So,Peter R. Stanfield 대한생리학회-대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba²⁺ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K⁺ currents in the absence and presence of externally applied Ba²⁺. Wild type Kir2.1 was blocked by externally applied Ba²⁺ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba²⁺ blockage. The estimated K_d(0) was 108ՌM for Kir2.1 wild type, 124ՌM for a concatameric WT-Y145V dimer, 109ՌM for a WT-Y145L dimer, and 267ՌM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba²⁺, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba²⁺ and a decrease in the on transition rate constant (k_on). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba²⁺ blockage in Kir 2.1.

Intrinsic Gating in Inward Rectifier Potassium Channels (Kir2.1) with Low Polyamine Affinity Generated by Site Directed Mutagenesis

So, I.,Ashmole, I.,Soh, H.,Park, C.S.,Spencer, P.J.,Leyland, M.,Stanfield, P.R. The Korean Society of Pharmacology 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.3

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for $K^+$, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of $P_{open}$ appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low $pH_i$. The effective valency for spermine is reduced from $3.09{\pm}0.07$ in wild type to $1.95{\pm}0.09$ in D172H at $pH_i$ 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

Intrinsic Gating in Inward Rectifier Potassium Channels (Kir2.1) with Low Polyamine Affinity Generated by Site Directed Mutagenesis

I So,I Ashmole,H Soh,CS Park,PJ Spencer,M Leyland,PR Stanfield 대한생리학회-대한약리학회 2003 The Korean Journal of Physiology & Pharmacology Vol.5 No.1

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for K<SUP>+</SUP>, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of <I>P</I><SUB>open</SUB> appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low pH<SUB>i</SUB>. The effective valency for spermine is reduced from 3.09⁑0.07 in wild type to 1.95⁑0.09 in D172H at pH<SUB>i</SUB> 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External $Ba^{2+}$

Lee, Young-Mee,Thompson, Gareth A.,Ashmole, Ian,Leyland, Mark,So, In-Suk,Stanfield, Peter R. The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.

A comparison between DASL and Affymetrix on probing the whole-transcriptome

정재식,Robert Audet,Jenny Chang,Helen Wong,Scooter Willis,Brandon Young,Susan Edgerton,Ann Thor,George Sledge,Renata Duchnowska,Jacek Jassem,Krzysztof Adamowicz,Brian Leyland-Jones,Changyu Shen 한국통계학회 2016 Journal of the Korean Statistical Society Vol.45 No.1

Whole-transcriptome microarray analysis has become a popular strategy to study geneexpression in cancer, exploiting the largely available formalin-fixed paraffin-embedded (FFPE) sample resources. However, there have been relatively few comparative studies evaluating the performance of the different gene-expression array platforms. We compared two commonly used whole-transcriptome microarray platforms: Illumina human whole genome cDNA-mediated annealing, selection extension and ligation (DASL) beadchip and Affymetrix U133 Plus2 GeneChip (Affymetrix). Gene expression data based on both platforms were collected on the same total RNA extracted from FFPE tissue samples of 221 advanced breast cancer patients. Correlations between two platforms were assessed using Pearson and Spearman correlation coefficients (CCs). For both platforms we also assessed coefficient of variation, which measures relative dispersion. Finally, we compared the applicability of DASL and Affymetrix for classification of breast cancer molecular subtypes using the PAM50 classifiers. Overall, there was a statistically significant, positive gene- and patient-wise correlation between the two platforms, with stronger relationship for patient-wise CC. The relative dispersion was smaller in DASL compared to Affymetrix. The consistency in subtype classification for both microarray platforms was weak (63%). We observed weak, yet positive correlation between two platforms and different magnitudes of correlations were observed according to the metrics used.