Calcium-dependent, slowly inactivating potassium currents in cultured neurons of rat neocortex

Abstract

Slowly inactivating outward currents were examined in neurons from rat anterior cortex dissociated at postnatal day 1 and recorded after 7–48 days in vitro by the use of whole-cell patch-clamp technique, in the presence of 0.5–0.8 μM tetrodotoxin (TTX), 50 μM carbachol and 1–5 mM CsCl₂. Experiments were often carried out in the additional presence of 1–5 mM CsCl₂, which blocks the anomalous, inwardly rectifying I _Q, the fast Ca ⁺₂ -dependent K⁺ current (I _C), and 50 μM carbachol, which depresses the I _M current. These currents were evoked by depolarizing steps to -40+-5 mV from a conditioning hyperpolarization to -110+-10 mV. Their sensitivity to elevation from 2.5 to 12.5 mM in extracellular K⁺ concentration, together with their sensitivity to 5–15 mM tetraethylammonium, suggests that they are mainly carried by K⁺ ions. Their activation and inactivation curves show that the threshold for activation is -65 mV, that their inactivation is achieved at -75 mV and that potentials more negative than -120 mV are needed to abolish it. The time-dependence of de-inactivation gives a maximal current amplitude for conditioning hyperpolarizations of 2 s and is best described by a monoexponential function with a time constant of 0.7 s. Slow, transient K⁺ currents were depressed by low doses of 4-aminopyridine (30–100 μM), which indicates the occurrence of an I _D-type component in the recorded K⁺ currents. No slowly declining K⁺ current was expressed when a recording solution containing 10 mM 1,2-bis(2-aminophenoxy)ethane-N, N,N′-N′-tetraacetic acid (BAPTA), instead of 1–5 mM BAPTA, was used. When recorded without Ca²⁺ chelator in the pipette, slowly declining K⁺ currents were blocked by bath-applied 40–50 μM BAPTA-aminoethoxy, revealing a large-amplitude, rapidly inactivating outward current. This residual component is insensitive to 50 μM 4-aminopyridine and may include a current more related to the I _A-type. Our data provide evidence that, in cultured cortical neurons from rat, the expression of an I _D-like K⁺ current is highly dependent on internal Ca²⁺ concentration.