| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
Single Cl and non-inactivating K channel currents were easily recorded in inside-out membrane patches in cultured Drosophila neurons. However, single Na channel currents were rarely observed, probably due to the low density of the channel in the culture condition. Here I focused on demonstrating a novel type of Cl channel that is blocked by commonly used buffering agents, HEPES, and on characterizing the permeability and gating properties of the non-inactivating K channels, compared with a mutant in which delayed rectifier K channel defect of Drosophila muscle has been proposed.
(1)The Cl channel has a unitary conductance of 35 pS, and stays at open states for tens of minutes with occasional interruptions by short closing transitions. When HEPES and MOPS (>1mM) were applied to the cytoplasmic face of membrane, it caused flickering within single openings of the Cl channel. A close inspection of records in HEPES has revealed that the open Cl channel has several subconductance levels, some
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of which are hardly detectable in the absence of HEPES. These results indicate that HEPES and MOPS act as an open channel blocker to Cl channels, and that Cl channels appear to function as a complex of multiple protochannels.
(2)The slope conductance of single non-inactivating K channel was estimated to 17.6 <-!+> 3.7pS when the cytoplasmic side of the inside-out membrane patch was perfused with solution containing 145 mM <K^+> . The single channel conductance was temperature-sensitive, with the Q10 of 1.44 between 10 and 20゜C. The conductance ratio of the channel for these cations was: <K^+> (1)> <NH_4^+> (0.53)> <Rb^+> (0.47)> <Na^+> (0.44). The open time distribution was fitted by a single exponential function, whereas closed time distribution required a three exponential fit. A sequential activation scheme with three closed states and one open state was proposed for the non-inactivating K channel of Drosophila. All transition rates between these states appeared to be voltage-dependent. Permeability and gating properties of single non-inactivating K channel currents in neurons of mutant eag, which is known to have defects in the delayed rectifier K channels of Drosophila muscle, were indistinguishable from those recorded from wild type neurons. Less
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