Then, PE-labeled mouse anti-human DR4 or DR5 mAb was added

n the same protein preparation extracted from HL-1 cells expressing hBK, we detected the voltage-gated K+ channels KV4.3, KV7.1 and KV11.1 that contribute to the repolarization phase of the AP. Only Kv11.1, detected as doublet immunoreactive bands at its predicted molecular weight of ~127 KD, showed a slight but significant decrease in expression when either both bands or the single upper band were analyzed. Notably, this loss of Kv11.1 protein in +hBK cells would be predicted to prolong APD, rather than contribute to shortening of APD after hBK transfection as a confounding effect. Western blots did not reveal changes in expression of the pore-forming subunits of the voltage-gated Na+ channel, which mediates the upstroke of the AP in HL-1 cells, or of the L-type Ca2+ channel, which is a critical contributor to the cardiac APD. hBK-transfected cells exhibit BK current HL-1 cells transfected with Null or +hBK plasmids were subjected to the whole-cell voltageclamp mode to record macroscopic membrane currents, and thereby identify functional BK channels. Unless otherwise noted, the predicted free-Ca2+ concentration in the pipette solution dialyzing the cells was buffered to 300 nM. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740312 This free-Ca2+ concentration is above normal resting levels in cardiac myocytes, but below levels of free-Ca2+ in contracting myocytes; nanomolar free-Ca2+ concentrations are adequate to activate BK channels when present. Fig 3 shows typical families of currents elicited from a holding potential of -70 mV with 500 ms pulses to potentials between -80 and +60 in 10 mV increments in Null and +hBK transfected HL-1 cells. The +hBK-transfected cells showed a significant increase in outward current densities at membrane potentials positive to +20 mV compared to the Nulltransfected cells, suggesting the expression of more functional K+ channels. The “noisy” outward currents at more positive potentials are typical of K+ currents mediated by BK 5 / 17 BK Channels In HL-1 Cells Shorten Action Potential Duration Fig 1. HL-1 Cell Immunofluorescence. Examples of HL-1 cells transfected with either +hBK plasmid bicistronically expressing mCherry and Flagtagged human BK gene or Null plasmid with mCherry only. A, D) mCherry fluorescence; B, E) anti-Flag staining in green; C, F) merged images. Scale bar, 20 m. doi:10.1371/journal.pone.0130588.g001 6 / 17 BK Channels In HL-1 Cells Shorten Action Potential Duration Fig 2. Characterization of ion channel proteins in HL-1 cells transfected with hBK. A) Examples of Western PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741685 blots comparing ion channel expression levels between HL-1 cells transfected with Null or +hBK for 48 h. Pore-forming -subunits of voltage-gated K+ channels native to HL-1 cells and voltage-gated Na+ and L-type Ca2+ channels were probed. Molecular mass markers for each blot are indicated on the right AZ-3146 column. B) Density of immunoreactive bands were averaged from 5 separate HL-1 cell cultures transfected with Null or +hBK. = significant difference from Null, P<0.05. See S1 Text and S1 Fig for complete Western blots of the data described in this Fig. doi:10.1371/journal.pone.0130588.g002 channels, which mediate large single-channel amplitudes at positive voltages. To show that this increased outward current was mediated by BK channels, iberiotoxin a specific inhibitor of BK channels was added to the patch-clamp chamber. Application of 100 nM IBTX did not significantly reduce the amplitude of outward currents in Null transfected cells, but eliminated noisy outward curren