voltage clamp method
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1995 ◽  
Vol 269 (2) ◽  
pp. H556-H564 ◽  
Author(s):  
T. Yamane ◽  
T. Furukawa ◽  
M. Hiraoka

The blocking action of 4-aminopyridine (4-AP) on the cloned K+ channel Kv1.5 expressed in Xenopus oocytes was studied using the two-microelectrode voltage-clamp method. Application of 4-AP to the bath solution reversibly suppressed the expressed current in a voltage- and concentration-dependent manner decreasing with membrane depolarization and with a half-maximal inhibitory concentration of 0.14 mM (at +40 mV). Both block and unblock occurred mainly during a depolarization when channels were activated. With successive depolarizations, 4-AP decreased not only the peak amplitudes of the current in successive pulses, but also the current during a depolarization. Upon washout of 4-AP, the current recovered with successive depolarizations, whereas no recovery of the current was noted in the absence of depolarizations. The extent of block markedly increased with alkalization of the external solution and decreased with acidification. External application of 4-amino-pyridine methiodide, a charged form of a quaternary 4-AP derivative, did not affect the current, but internal application markedly suppressed the current, indicating the drug gained access to the channel from the cytoplasmic side. These data suggest that 4-AP crosses the membrane in its uncharged form and acts from inside of the cell in its charged form, resulting in block of the channels with higher affinity to the open state.


1993 ◽  
Vol 265 (5) ◽  
pp. R990-R1000 ◽  
Author(s):  
G. Samman ◽  
M. Ohtsuyama ◽  
F. Sato ◽  
K. Sato

In isolated rhesus eccrine clear cells, regulatory volume decrease (RVD) occurs after osmotic swelling. RVD was completely inhibited by 1 mM quinidine, 200 nM charybdotoxin, 1 mM diphenylamine-2-carboxylic acid (DPC), or 0.1 mM 4-nitro-2(3-phenylpropyl-amino)benzoate. RVD was also inhibited in Ca(2+)-free medium by vinblastine (antimicrotubular agent), N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7), or 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Valinomycin reversed quinidine- and DIDS-induced inhibition of RVD but not the inhibition caused by Ca(2+)-free medium, DPC, vinblastine, or W-7. The cytosolic free Ca2+ concentration, as determined by the fura 2 method, increased from 220 nM in the control to 435 nM during RVD. Activation of both K+ and Cl-currents was also directly demonstrated with the whole cell current-voltage clamp method. DIDS inhibited swelling-induced K+, but not Cl-, currents and depolarized the membrane potential during RVD, further supporting the notion that DIDS inhibited swelling-activated K+, but not Cl-, pathways. We conclude that the observed RVD is mediated by the activation of conductive Ca(2+)-dependent K+ and Cl- pathways.


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