Constitutively active G-protein-gated inwardly rectifying K+ channels in dendrites of hippocampal CA1 pyramidal neurons

Abstract

A diversity of ion channels contributes to the active properties of neuronal dendrites. From the apical dendrites of hippocampal CA1 pyramidal neurons, we recorded inwardly rectifying K+ channels with a single-channel conductance of 33 pS. The inwardly rectifying K+ channels were constitutively active at the resting membrane potential. The amount of constitutive channel activity was significantly larger in the apical dendrites than in the soma. Activities of these inwardly rectifying K+ channels were inhibited by Ba2+ (200 microM) and tertiapin (10 nM), both of which are believed to block G-protein-coupled inwardly rectifying K+ (GIRK) channels. Intracellularly applied GTPgammaS (20 microM) during dual dendritic recordings significantly increased constitutive channel activity. Baclofen (20 microM), an agonist for the G-protein-coupled GABA(B) receptor, also significantly increased the level of channel activity. Therefore, these channels are GIRK channels, which are constitutively active at rest in the apical dendrites of CA1 pyramidal neurons and can be further activated via G-protein-coupled neurotransmitter receptors.

Figure 1.

Single-channel analysis of inwardly rectifying K⁺ channel(s) and channel distribution along the dendrites. A, Single inwardly rectifying K⁺ channel(s) recorded at 200 μm from the soma. Inset, An expanded stretch of record showing an idealized trace (smooth line; generated with HMMs in TAC software) overlaying with the original trace. Calibration: 1 pA, 20 ms. B, Current-voltage plot under control (Ctrl) condition (γ = 33 pS) in the presence of GTPγS (γ = 33 pS) or the presence of baclofen (γ = 31 pS). Some error bars are smaller than the symbols. Single-channel conductance was determined as the slope of the best-fitted line in the linear I-V range. C, Open probability plotted against membrane potential. D, The level of constitutive channel activity (measured at resting potential) was plotted against distance from the soma. The amount of channel activity was significantly larger in the dendrites compared with the soma. Recordings were binned for every 50 μm. Soma, NP_o = 0.0038 ± 0.0012 (n = 5); 50 μm, NP_o = 0.0085 ± 0.0051 (n = 6); 100 μm, NP_o = 0.027 ± 0.0066 (n = 4); 150 μm, NP_o = 0.031 ± 0.012 (n = 5); 200 μm, NP_o = 0.018 ± 0.0053 (n = 4); 250 μm, NP_o = 0.031 ± 0.010 (n = 5); 300 μm, NP_o = 0.024 ± 0.0074 (n = 8). ^*p < 0.05 and ^**p < 0.01 compared with the soma. s.o., Stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum; s.l-m, stratum lacunosum-moleculare. Error bars represent SEM.

Figure 2.

Pharmacology of the inwardly rectifying K⁺ channels. A, Trace examples with control solutions, Ba²⁺, or tertiapin in the recording pipette. The membrane was held at resting potentials. B, Both Ba²⁺ and tertiapin inhibited constitutive activities of the inwardly rectifying K⁺ channels. Control (Ctrl), NP_o = 0.031 ± 0.007, 218 ± 9 μm from the soma (n = 11); 200 μm Ba²⁺, NP_o = 0.011 ± 0.003, 228 ± 7 μm from the soma (n = 9); 2 mm Ba²⁺, NP_o = 0.003 ± 0.001, 258 ± 20 μm from the soma (n = 4); 10 nm tertiapin, NP_o = 0.015 ± 0.004, 215 ± 5 μm from the soma (n = 10); 50 nm tertiapin, NP_o = 0.005 ± 0.001, 224 ± 8 μm from the soma (n = 8); 2 μm tertiapin, NP_o = 0.001 ± 0.001, 223 ± 8 μm from the soma (n = 3). ^*p < 0.05 and ^**p < 0.01 compared with control. Error bars represent SEM.

Figure 3.

Activation of inwardly rectifying K⁺ channels by intracellularly applied GTPγS. A, Position of a whole-cell and a cell-attached electrode. The whole-cell electrodes contained 0, 20, or 200 μm GTPγS and bis-fura-2. The picture was taken after the experiment, showing the dendrite filled with bis-fura-2. B, Trace examples with no GTP or GTPγS in the whole-cell electrode, with or without Ba²⁺ in the cell-attached electrode. The membrane was held at resting potentials. C, Applying 20 μm GTPγS into the cell significantly increased the level of channel activity. GTPγS (200 μm) did not further increase channel activity. Ba²⁺ (200 μm) reduced channel activation with GTPγS by ∼67%. No GTP, NP_o = 0.013 ± 0.006, 206 ± 8 μm from the soma (n = 5); 20 μm GTPγS, NP_o = 0.127 ± 0.033, 206 ± 7 μm from the soma (n = 6); 200 μm GTPγS, NP_o = 0.118 ± 0.026, 213 ± 8 μm from the soma (n = 4); 20 μm GTPγS with 200 μm Ba²⁺, NP_o = 0.042 ± 0.015, 220 ± 16 μm from the soma (n = 4). ^*p < 0.05 compared with control; ^#p < 0.05 compared with 20 μm GTPγS. Error bars represent SEM.

Figure 4.

Stimulating GABA_B receptors activated GIRK channels in distal dendrites. A, Trace examples with control solution or 20 μm baclofen in the recording pipette. The membrane was held at resting potentials. B, In 10 of 19 recordings, stimulation of GABA_B receptors by baclofen led to increased GIRK channel activity. Control (Ctrl), NP_o = 0.024 ± 0.007, 303 ± 4 μm from the soma (n = 8); 20 μm baclofen, NP_o = 0.119 ± 0.017, 304 ± 9 μm from the soma (n = 10); NP_o = 0.022 ± 0.004, 304 ± 8 μm from the soma (n = 9). ^**p < 0.01 compared with control. Error bars represent SEM.