Barros F, Katz G M, Kaczorowski G J, Vandlen R L, Reuben J P
Proc Natl Acad Sci U S A. 1985 Feb;82(4):1108-12. doi: 10.1073/pnas.82.4.1108.
To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.
为了分离GH3大鼠垂体细胞中的内向Ca2+电流，必须抑制内向Na+电流以及两种外向K+电流，即瞬态电压依赖性电流（IKV）和缓慢上升的Ca2+激活电流（IKCa）。通常通过用Cs+替代细胞内K+来阻断这些外向电流，从而揭示出持续的内向电流。在细胞内存在K+的情况下，可使用季铵离子选择性阻断任一K+电流。当IKCa和Na+电流被阻断时，将膜电位（Vm）从-60 mV步进到0 mV所引发的净电流首先是内向的，然后变为外向并在10 - 30毫秒内达到峰值，最后又再次变为内向。在这种情况下，在电压阶跃的整个持续时间内IKV和Ca2+电流都应存在，但在向外电流峰值时未检测到Ca2+电流。也就是说，向外电流峰值与Vm的关系图是单调的，并且不受尼索地平或低细胞外Ca2+的影响，而如果存在Ca2+电流，预期会受到影响。然而，在电流峰值以外的其他时间的类似关系图不是单调的，并且会被尼索地平或低Ca2+改变（即内向电流减小且关系图变为单调）。当通过将Vm保持在-30 mV使K+通道首先失活时，将Vm步进到0 mV时总是会观察到持续的Ca2+电流。此外，用Ba2+替代Ca2+会导致IKV被阻断，并且该电流的抑制会导致具有方波动力学的内向Ba2+电流。这些数据表明，在向外IKV峰值时Ca2+电流被完全抑制，并且随着瞬态K+电流因通道失活而下降，Ca2+电导逐渐去抑制。这表明在GH3细胞中Ca2+通道受IKV调节。
