Highly co-operative Ca2+ activation of intermediate-conductance K+ channels in granulocytes from a human cell line.
1. To study Ca(2+)-activated K+ currents in dimethyl sulphoxide (DMSO)-differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-L-phenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)-activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nM [Ca2+]i, and a highly co-operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin-sensitive, intermediate-conductance Ca(2+)-activated K+ channel in HL-60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co-operativity and voltage independence) are similar to the Ca2+ activation of the apamin-sensitive small-conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL-60 granulocytes.
Varnai
P
P
Infectious Diseases Division, University Hospital, Geneva, Switzerland.
Demaurex
N
N
Jaconi
M
M
Schlegel
W
W
Lew
D P
DP
Krause
K H
KH
D001030:Apamin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D018999:Charybdotoxin; D003600:Cytosol / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D015221:Potassium Channels / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D012604:Scorpion Venoms / Q000494:pharmacology
D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D001030:Apamin; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium; D004121:Dimethyl Sulfoxide