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 108micrometer for Kir2.1 wild type, 124micrometer for a concatameric WT-Y145V dimer, 109micrometer for a WT-Y145L dimer, and 267micrometer 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.
Animals ; Cricetinae ; Cricetulus ; Cytoplasm ; Female ; Kinetics ; Membranes ; Ovary ; Patch-Clamp Techniques ; Potassium Channels ; Potassium Channels, Inwardly Rectifying

Recent experience with pandemic influenza A(H1N1)pdm09 highlighted the importance of global surveillance for severe respiratory disease to support pandemic preparedness and seasonal influenza control. Improved surveillance in the southern hemisphere is needed to provide critical data on influenza epidemiology, disease burden, circulating strains and effectiveness of influenza prevention and control measures. Hospital-based surveillance for severe acute respiratory infection (SARI) cases was established in New Zealand on 30 April 2012. The aims were to measure incidence, prevalence, risk factors, clinical spectrum and outcomes for SARI and associated influenza and other respiratory pathogen cases as well as to understand influenza contribution to patients not meeting SARI case definition.All inpatients with suspected respiratory infections who were admitted overnight to the study hospitals were screened daily. If a patient met the World Health Organization’s SARI case definition, a respiratory specimen was tested for influenza and other respiratory pathogens. A case report form captured demographics, history of presenting illness, co-morbidities, disease course and outcome and risk factors. These data were supplemented from electronic clinical records and other linked data sources.Hospital-based SARI surveillance has been implemented and is fully functioning in New Zealand. Active, prospective, continuous, hospital-based SARI surveillance is useful in supporting pandemic preparedness for emerging influenza A(H7N9) virus infections and seasonal influenza prevention and control.