Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): effects of K+, Na+ and Mg2+.
Patch-clamp techniques were used to study the permeability to ions of an ATP-sensitive channel in membranes from the pancreatic B-cell line (RINm5F). With patches in the outside-out configuration, the I-V curves for different Na+-K+ mixtures in the bath and 140 mM K+ in the pipette were almost linear, and crossed the zero-current axis at voltages that indicated a variable permeability ratio. When K+ was added symmetrically, the plot of the conductance vs. K+ activity exhibited saturation, with a Gmax of about 160 pS and a half-maximal activity of 216 mM. The I-V behavior for different K+-Na+ mixtures in the bath could be accurately described with a model based on Eyring theory, assuming two sites and one-ion occupancy. For K+, the dissociation constants (KK) of the two sites were 290 and 850 mM, the lower value pertaining to the site close to the intracellular medium. In experiments with inside-out patches, both Na+ and Mg2+, when present in the bath, induced a voltage-dependent block of the outward current. Fitting the data with the model suggested that for these ions only one of the two sites binds significantly, the corresponding dissociation constants being (mM): 46 for Na+ and 34 for Mg2+. Blocking by Na+ and Mg2+ may account for the low outward current seen in intact cells. This hypothesis is consistent with the observation that such current is further reduced by addition of 2,4-DNP, since metabolism inhibitors are expected to lower the ATP level, thereby liberating Mg2+ from the Mg2+-ATP complex, as well as inducing accumulation of Na+ by decreasing the rate of the Na+-K+ pump.
Ciani
S
S
Department of Physiology, Ahmanson Laboratory of Neurobiology, University of California, Los Angeles.
Ribalet
B
B
D019297:2,4-Dinitrophenol; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004140:Dinitrophenols / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology* / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology* / Q000502:physiology; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism
D004140:Dinitrophenols; D007328:Insulin; D015221:Potassium Channels; D015222:Sodium Channels; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D008274:Magnesium; D019297:2,4-Dinitrophenol; D011188:Potassium