cell suspension transferred to a glass bottom perfusion chamber on the stage of an inverted microscope. The chamber was perfused intermittently with control HBSS of the following composition: 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 5.5 dextrose. Patch electrodes were filled with a K+-aspartate solution consisting of: 115 KOH, 115 DL-aspartic acid, 30 KCl, 1 EGTA, 10 HEPES, 1 MgATP and 10 nM or 300 nM free PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741226 CaCl2. All experiments were performed at room temperature. Electrophysiological measurements were made using conventional techniques and methods previously described. Data obtained in response to voltage or current pulses were low pass filtered by either analog or digital filters at 1 to 5 kHz and sampled at 2 to 10 kHz as appropriate for channel kinetics. Patch pipettes were made from borosilicate glass pulled on a Brown-Flaming P77 puller and fire polished to have a final resistance of 35 MO. Capacitance and series resistance compensation were used to improve the quality of the voltage clamp and reduce associated artifacts. Whole-cell currents were elicited from a holding potential of -70 mV with 500 ms pulses to potentials between -60 and +60 in 10 mV increments. P/4 protocols were used to cancel leak and capacitance currents when pulse protocols were applied. Action potentials were recorded in current-clamp mode by applying a 1 ms pulse of 5 pA at 
0.5 Hz following a 100 ms pulse of -5 pA to elicit an anode break excitation. Statistical Analysis All experiments were replicated 5 to 10 times. Measured or LOXO-101 calculated parameters are reported as mean SEM. Currents were normalized to cell size by dividing by capacitance. All experiments were analyzed by unpaired Student’s t-test, except for comparisons of I-V curve 4 / 17 BK Channels In HL-1 Cells Shorten Action Potential Duration amplitudes, which were analyzed using analysis of variance with a Tukey post-hoc test. The criterion for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740492 a significant difference was a P value less than 0.05. Results Expression of hBK and other ion channels in HL-1 Cells HL-1 cells were transfected for 48 to 72 hr with either +hBK plasmid bicistronically expressing mCherry and Flag-tagged human BK gene or Null plasmid with mCherry only. Transfected cells identified by red fluorescence were used in all subsequent patch clamp experiments. As expected, +hBK-transfected cells were positive for anti-Flag staining but not Null-transfected cells. Changes in the expression of one ion channel type can alter the abundance of other ion channels, and this “remodeling” process includes cardiac myocytes that show a propensity to adapt their ion channel profiles. For example, deletion of one or more K+ channel genes may affect the expression levels of other K+ channels in cardiac myocytes in vivo. Also, changes in the expression level of K+ channels may alter the cell resting membrane potential, which subsequently can influence the abundance of L-type Ca2+ channels. Based on these and other reports, we employed Western blotting to define the effect of +hBK transfection on the expression of ion channels involved in generating APs in HL-1 cells. Our intent was to ensure that any shortening of APD attributed to hBK expression was not related to an unintended increase of repolarizing K+ channels or to a loss of depolarizing currents through voltage-gated Na+ channels or L-type Ca2+ channels. First, we showed in Western blots using anti-Flag antibody that exogenous human BK subunit was robustly expressed in +hBK cells. I
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