Ca2+‐inhibited non‐inactivating K+ channels in cultured rat hippocampal pyramidal neurones

Abstract

Whole‐cell perforated‐patch recording from cultured CA1‐CA3 pyramidal neurones from neonatal rat hippocampus (20‐22 °C; [K⁺]_o= 2.5 mM) revealed two previously recorded non‐inactivating (sustained) K⁺ outward currents: a voltage‐independent ‘leak’ current (I_leak) operating at all negative potentials, and, at potentials ≥−60 mV, a time‐ and voltage‐dependent ‘M‐current’ (I_K(M)). Both were inhibited by 1 mM Ba²⁺ or 10 μM oxotremorine‐M (Oxo‐M). In ruptured‐patch recording using Ca²⁺‐free pipette solution, I_leak was strongly enhanced, and was inhibited by 1 mM Ba²⁺ but unaffected by 0.5 mM 4‐aminopyridine (4‐AP), 1 mM tetraethylammonium (TEA) or 1‐10 nM margatoxin. Single channels underlying these currents were sought in cell‐attached patch recordings. A single class of channels of conductance ≈7 pS showing sustained activity at resting potential and above was identified. These normally had a very low open probability (P_o < 0.1), which, however, showed a dramatic and reversible increase (to about 0.9 at ≈0 mV) following the removal of Ca²⁺ from the bath. Under these (Ca²⁺‐free) conditions, single‐channel P_o showed both voltage‐dependent and voltage‐independent components on patch depolarization from resting potential. The mean activation curve was fitted by a modified Boltzmann equation. When tested, all channels were reversibly inhibited by addition of 10 μM Oxo‐M to the bath solution. The channels maintained their high P_o in patches excised in inside‐out mode into a Ca²⁺‐free internal solution and were strongly inhibited by application of Ca²⁺ to the inner face of the membrane (IC₅₀= 122 nM); this inhibition was observed in the absence of MgATP, and therefore was direct and unrelated to channel phosphorylation/dephosphorylation. Channels in patches excised in outside‐out mode were blocked by 1 mM Ba²⁺ but were unaffected by 4‐AP or TEA. Channels in cell‐attached patches were inhibited after single spikes, yielding inward ensemble currents lasting several hundred milliseconds. This was prevented in Ca²⁺‐free solution, implying that it was due to Ca²⁺ entry. The properties of these channels (block by internal Ca²⁺ and external Oxo‐M and Ba²⁺, and the presence of both voltage‐dependent and voltage‐independent components in their P_o/V relationship) show points of resemblance to those expected for channels associated with both I_leak and I_K(M) components of the sustained macroscopic currents. For this reason we designate them K_sust (‘sustained current’) channels. Inhibition of these channels by Ca²⁺ entry during an action potential may account for some forms of Ca²⁺‐dependent after‐depolarization. Their high sensitivity to internal Ca²⁺ may provide a new, positive feedback mechanism for cell excitation operating at low (near‐resting) [Ca²⁺]_i.