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Avoid Ku-0059436 Challenges And How To Identify Any Of Them

Added: (Fri Feb 02 2018)

Pressbox (Press Release) - CHO cells with at least 80% confluence were transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with 3 ��g of TWIK-1 plasmids and 1 ��g of pEGFP plasmids and studied 24?h later. GFP expression was used to identify effectively transfected CHO cells. Whole-cell patch-clamp recordings were performed and data www.selleckchem.com were analyzed, as described previously ( 7). Briefly, whole-cell currents of TWIK-1 K+ channels in transfected CHO cells were recorded with a standard 2.2?s voltage ramp from ?140?mV to +80?mV each 15 s. In Figs. 1 and 2, whole-cell currents at +80?mV in Na+-based bath solutions with 5?mM [K+]o are <250?pA. The pipette solution contained 140?mM KCl, 1?mM MgCl2, 10?mM EGTA, 1?mM K2-ATP, and 5?mM HEPES (pH7.4). The Na+-based bath solution contained 135?mM NaCl, 5?mM KCl, 2?mM CaCl2, 1?mM MgCl2, and 10?mM HEPES (pH7.4). The total concentration of Na+ and K+ in bath solutions is 140?mM so bath solutions with various [K+]o were obtained by exchanges of equimolar K+ and Na+. The Rb+ or NH4+-based bath solutions were obtained by replacing extracellular Na+ by equimolar monovalent cations. We have previously shown that in the transfected CHO cells, by which large TWIK-1 K+ currents are measured, TWIK-1 K+ channels change ion selectivity and conduct inward leak Na+ currents in lowered extracellular K+ concentrations ([K+]o) (7). Thus, we investigated whether silent Pomalidomide TWIK-1 K+ channels heterologously Ku-0059436 solubility dmso expressed in CHO cells exhibit the same characteristic in lowered [K+]o, become permeable to extracellular Na+, and conduct inward Na+ currents. Consistent with previous reports (7, 8?and?11), TWIK-1 K+ channels do not produce measurable currents in most of the transfected CHO cells in Na+-based bath solutions with physiological [K+]o. Whole-cell currents recorded at ?140?mV and +80?mV are ?95 �� 6?pA and 188 �� 19?pA (n = 4), respectively (black line, Fig.?1B), within the range of background noises. However, removing 5?mM K+ in the bath solution converted silent TWIK-1 K+ channels into nonselective cation channels that conducted large inward Na+ and outward K+ currents with a reversal potential of ?15.6 �� 1.5?mV (n = 4) (red line, Fig.?1B) and a Na+ to K+ relative permeability of ?0.53. It took ?10?min for TWIK-1 channels to complete the selectivity switch process. Moreover, whole-cell currents recorded in ?140?mV (?843?�� 191?pA) and +80?mV (637 �� 119?pA) were significantly increased by 8.8-fold and 3.3-fold, respectively. These results are consistent with previous observations in those transfected CHO cells that large TWIK-1 K+ currents are detected ( 7). In contrast, these results were not seen in CHO cells transfected with GFP alone (n = 20; Fig.?1A). Such an amount of inward Na+ and outward K+ currents in 0?mM [K+]o indicates that TWIK-1 K+ channels are expressed on the surface of transfected CHO cells.

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