| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
To establish ion conducting properties of the N-type Ca^<2+> channel, we examined a recombinant N-type Ca^<2+> channels expressed in baby hamster kidney cells, using a conventional whole-cell patch-clamp technique.The recombinant N-type Ca^<2+> channel, composed of the alpha_<1B>, alpha_<1*> and beta_<1*> subunits, displayed high-voltage-activated Ba^<2+> currents, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA.In the presence of 110mM Ba^<2+>, the unitary current showed a slope conductance of 18.2pS, characteristic of N-type channels. Ca^<2+> and Sr^<2+> resulted in smaller ion fluxes than Ba^<2+>, with the ratio 1.0 : 0.72 : 0.75 of maximum conductance in current-voltage relationships of Ba^<2+>, Ca^<2+> and Sr^<2+> currents, respectively. In mixtures of Ba^<2+> and Ca^<2+>, where the Ca^<2+> concentration was steadily increased in place of Ba^<2+>, with the total concentration of Ba^<2+> and Ca^<2+> held constant at 3mM, the current amplitude went throu
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gh a clear minimum when 20% of the external Ba^<2+> was replaced by Ca^<2+>.This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously.Using an external solution containing 110mM Na^+ without polyvalent cations, inward Na^+ currents were evoked by test potentials more positive than -5OmV.These currents were activated and inactivated in a kinetic manner similar to that of Ba^<2+> currents.Application of inorganic Ca^<2+> antagonists blocked Ba^<2+> currents through N-type channels in a concentration-dependent manner.The rank order of inhibition was La^<3+> <greater than or equal> Cd^<2+> * Zn^<2+> * Ni^<2+> <greater than or equal> Co^<2+>.When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La^<3+> and Cd^<2+>, and subsequent channel reblocking was observed.The measured rate (2x10^8M^<-1>s^<-1>) of reblocking approached the diffusion-controlled limit.These results suggest that N-type Ca^<2+> channels share general features of a high affinity ion-binding site with the L-type Ca^<2+> channel, and that this site is easily accessible from the outside of the channel pore.
channel calcium patch-clamp omega-conotoxin-GVIA Less
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