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Ten
microliters of virus (which was concentrated Sodiulm-Specific Membrane Channels of Frog Skin Are Pores:
100-fold by centrifugation and then suspended
in 50 mM tris-HCI, pH 7.3, 10 mM dithio- Current Fluctuations Reveal High Turnover
threitol, and bovine serum albumin, I mg/
ml) were incubated with 10 ,ul of 50 mM tris-
HCI, pH 7.3, 10 mM dithiothreitol, 0.06 percent Abstract. The reversible sodium transport blocker amiloride causes current fluc-
Triton X-100 for 10 minutes at 2C. A portion (20 tuations at the apical membrane of the outer stratum granulosum offrog skin. Their
I1A) of assay mixture was added, and the result-
ing 40-,ul reaction mixture was incubated for 60 power density spectra reveal that single transport sites translocate more than 106 So-
minutes at 37C. The reaction was stopped by dium ions per second, which indicates a pore mechanism. The density of open plus
the addition of 0.5 ml of 80 mM sodium pyro-
phosphate, 25 ,u of bovine serum albumin (5 mg/ amiloride-blocked pores is in the order of 108 pores per square centimeter of skin
ml), and 0.5 ml of 25 percent trichloroacetic acid
at 2C. Acid-insoluble material was collected on area with 60 millimolar sodium and 18 micromolar amiloride in the outer solution.
glass fiber filters, washed extensively with 10
percent trichloroacetic acid, dried, and dis-
solved in NCS tissue solubilizer; the radio- Carrier- and pore-mediated transport mined by the apical membrane of the
activity was then counted in toluene-based
scintillation fluid. The reaction mixtures with through biological membranes can be dis- stratum granulosum (4). The current
poly(rA) * oligo(dT)12_18 and poly(dA) * oligo
(dT)12-18 consisted of: 50 mM tris-HCI, pH
tinguished by the turnover number of in- component that did not pass the Na-spe-
7.3, 70 mM KCI, 10 mM dithiothreitol, 0.1 mM dividual transport sites (1). While a car- cific channels was determined as the cur-
each of deoxyriboadenosine triphosphate (dATP), rier molecule, which has to move rent flowing in the presence of 35 pM
deoxyriboguanosine triphosphate (dGTP),
deoxyribocytidine triphosphate (dCTP), 2.3 through the lipid phase of the membrane, amiloride, and was subtracted from the
pM 3H-labeled thymidine triphosphate (40,000 is not likely to transport more than 104 total current to obtain the transcellular
count/mnin per picomole), 20 j.g of poly(rA) or
poly(dA) per milliliter, 20 pg of oligo(dT)12_18 ions or molecules per second, the trans- Na current, INa-
per milliliter, and either 5 mM MgCl2 or 0.4
mM MnCI2. The reaction mixture containing port rate of a pore can be several orders Transepithelial voltage was clamped
poly(rC) oligo(dG)12-18 consisted of: 50 mM
tris-HCI, pH 7.3, 50 ,ug of actinomycin D
of magnitude larger (2). Applying this to 0 mv by a voltage clamp circuit with
that the plateau value, S0, of the power where ,8 = (A)m/(A)o is the unknown par- For (Na)0 = 60 mM, values in the range
spectrum decreases with increasing tition coefficient of amiloride between 0.3 to 0.5 pa were computed, which cor-
amiloride concentration while the corner the outer bulk solution and the space at respond to turnover numbers of 1 to 3 x
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