Exploring calcium channels using divalent cations and specific channel blockers in frog sympathetic neurons
Description
In the present study, distinctive interactions of divalent cations and specific blockers with calcium channels were exploited to probe several functional features related to permeation through calcium channels in frog sympathetic neurons In the first study, our experiments have focused on resolving a paradox why the whole-cell current has not been observed under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels are referred to as Ef-channels, and they are present in the membrane at a density greater than the channels that carry ∼90% of whole-cell current in low Ba2+, but Ef-current has not been detected in low Ba2+. Using Ca2+, instead of Ba2+ as the charge carrier, we recorded a possible E-type current in frog sympathetic neurons. The current contributed about 12% of total current at peak voltage in 3 mM Ca2+ , and increased at voltages more hyperpolarized to the peak, reaching ∼40% at -30 mV. The presence of Ef-current in 3 mM Ca2+ , suggests a potential role of Ef-channels in regulating calcium influx into sympathetic neurons In the second study, the effect of Ba2+ on the block of N-channels by o-conotoxin GVIA (oCGVIA) was investigated. oCGVIA has been reported to be an irreversible blocker of N-type calcium channels (Cav 2.2). Surprisingly, oCGVIA block was rapidly reversed in 0 Ba2+. The off-rate of oCGVIA from N-channels was dependent on [Ba2+]o, since at an intermediate concentration (3 muM Ba2+), current recovered with a tau of 64 +/- 16 s (n = 6), significantly slower than in 0 Ba2+ but faster than in 3 mM Ba2+ In the final study, we examined Ca2+ permeation through N-channels. Models of permeation through voltage-gated Ca2+ channels have been constructed based on L-channel data. Specifically, our goal was to determine if an apparent high affinity binding site underlies channel selectivity for Ca2+, and an apparent low affinity binding site supports Ca2+ conductance in N-channels. Our data confirmed that Ca2+ binding inside N-channels is involved in both Ca2+ selectivity and conductance. (Abstract shortened by UMI.)