Properties of single voltage-dependent K+ channels in dendrites of CA1 pyramidal neurones of rat hippocampus

Abstract

Voltage-dependent K(+) channels in the apical dendrites of CA1 pyramidal neurones play important roles in regulating dendritic excitability, synaptic integration, and synaptic plasticity. Using cell-attached, voltage-clamp recordings, we found a large variability in the waveforms of macroscopic K(+) currents in the dendrites. With single-channel analysis, however, we were able to identify four types of voltage-dependent K(+) channels and we categorized them as belonging to delayed-rectifier, M-, D-, or A-type K(+) channels previously described from whole-cell recordings. Delayed-rectifier-type K(+) channels had a single-channel conductance of 19 +/- 0.5 pS, and made up the majority of the sustained K(+) current uniformly distributed along the apical dendrites. The M-type K(+) channels had a single-channel conductance of 11 +/- 0.8 pS, did not inactivate with prolonged membrane depolarization, deactivated with slow kinetics (time constant 100 +/- 6 ms at -40 mV), and were inhibited by bath-applied muscarinic agonist carbachol (10 microm). The D-type K(+) channels had a single-channel conductance of around 18 pS, and inactivated with a time constant of 98 +/- 4 ms at +54 mV. The A-type K(+) channels had a single-channel conductance of 6 +/- 0.6 pS, inactivated with a time constant of 23 +/- 2 ms at +54 mV, and contributed to the majority of the transient K(+) current previously described. These results suggest both functional and molecular complexity for K(+) channels in dendrites of CA1 pyramidal neurones.

Figure 1. Variability of macroscopic K⁺ currents

All current traces (ensemble averages of 3–20 sweeps) shown in this figure were recorded from dendritic patches at 220 ± 3 μm from soma. Electrodes used in these recordings had uniform diameters of ∼1 μm. Minimum suction was applied during seal formation. K⁺ currents were activated by a voltage step from approximately −96 mV to +54 mV, lasting 400 ms. Both the amplitude and the waveform of the macroscopic currents were highly variable.

Figure 2. Single delayed-rectifier K⁺ channel recorded 200 μm from soma

Only one delayed-rectifier-type K⁺ channel was present in this recording. A, example traces of single-channel activity. The patch membrane was held at −98 mV. Voltage commands of 1000 ms were given to activate the channel. Channel activity increased with depolarization. Bottom trace is the ensemble average of recordings at +12 mV (average of 25 traces). Single exponential fitting to the ensemble average gave τ = 1.7 s for time-dependent inactivation. B, current amplitude histograms of recordings at noted potentials. The left peak in each histogram represents current level when the channel was closed, while the right peak in the histogram represents current level when the channel was open. C, amplitude scatter graph. Current amplitude of the open state is plotted against command potential. The line fit to the amplitude scatter graph gave single-channel conductance (γ) of 18 pS. D, voltage-dependent activation. Open probability of the channel was plotted against command potential. Activation can be fitted with a single Boltzmann equation. In this particular recording, half-activation voltage (V_½) was +5 mV; slope of activation (k) was 7.3. E, duration analysis. Duration histograms were constructed for both closed and open times at noted potentials. The histograms could be best fitted with two-component exponential distribution functions (continuous lines). Dotted lines mark the time constants of the second components of each fit (τ_c2 and τ_o2): V_m = −8 mV, τ_c2 = 54 ms, τ_o2 = 6 ms; V_m = +2 mV, τ_c2 = 21 ms, τ_o2 = 12 ms; V_m = +12 mV, τ_c2 = 6 ms, τ_o2 = 17 ms.

Figure 3. Recording of two different types of K⁺ channels in the same patch, 230 μm from soma

A, example traces of channel activities recorded with 0 Ca²⁺ in the recording pipette. Step commands of noted potentials (from a holding potential of −100 mV) were given to the patch. A delayed-rectifier K⁺ channel started to open at −20 mV, while the activities of a smaller conductance channel (M-type K⁺ channel) could be seen at −30 mV. Activities of both channels increased with depolarization. B, current amplitude histograms at noted potentials. At a membrane potential of −10 mV, the histogram has two distinct peaks. The left peak represents current level when both channels were closed, while the right peak represents current level when only the smaller channel alone was open. At this membrane potential, opening of the delayed-rectifier-type channel did not contribute a distinctive current peak to the histogram. At more depolarized potentials, the amplitude histograms have four peaks, representing current levels when both channels were closed, when the smaller channel alone was open, when the delayed-rectifier channel alone was open, and when both channels were open at the same time. C, amplitude scatter can be fitted with two lines each representing a conductance level. The delayed-rectifier channel in this recording (grey symbols) had a single-channel conductance (γ) of 19 pS. The M-type K⁺ channel had a γ of 10 pS. D, open probability of each channel was plotted against membrane potential. Activation of the delayed-rectifier channel was fitted with a single Boltzmann equation which gave V_½ = +9 mV and k = 7.5 (grey symbols). Activation of the M-type K⁺ channel was fitted with a single Boltzmann equation with V_½ = −6 mV and k = 16.9.

Figure 4. Duration analysis of an M-type channel

Analysis was performed on the M-type channel in Fig. 3. Both closed and open time histograms can be best fitted with single-component exponential distribution functions (continuous lines). Dotted lines mark the time constants of each fit (τ_c and τ_o). A, at V_m = −20 mV, τ_c = 54 ms, τ_o = 12 ms. B, at 0 mV, τ_c = 16 ms, τ_o = 15 ms. C, at V_m = +20 mV, τ_c = 8 ms, τ_o = 61 ms.

Figure 5. Time-dependent kinetics of the M-type K⁺ channels

A, the delayed-rectifier K⁺ channel and the M-type K⁺ channel have different inactivation properties. In response to a 15 s continuous voltage step to +20 mV (from a holding potential of −70 mV), the delayed-rectifier channel (upper trace, 210 μm from soma) inactivated within 3 s of the start of the step. The M-type channel (lower trace, 230 μm from soma, different patch) did not inactivate for 15 s with depolarization. B and C, deactivation and reactivation kinetics of the M-type K⁺ channel. Recordings were performed from a somatic patch with multiple K⁺ channels. The membrane was first depolarized to +18 mV (from −72 mV) for 10 s so that only the M-type K⁺ channel remained active. At the end of this 10 s depolarization, the membrane was hyperpolarized to −12, −42 or −72 mV for 1 s to deactivate the channel, after which the membrane was depolarized to +18 mV to reactivate the channel. Example single traces are shown on the left, averages are shown on the right. Dotted lines indicate the current level when the channel was closed. B, the M-type K⁺ channel deactivated with τ = 99 and 51 ms at −42 and −72 mV, respectively. Channel deactivation was not obvious at −12 mV as there were still appreciable channel activities at this voltage. C, from −12 mV, the partially deactivated M-type K⁺ channel reactivated with τ = 24 ms as the membrane was depolarized to +18 mV. From −42 mV, the deactivated channel reactivated with τ = 13 ms. From −72 mV, the step to +18 mV reactivated an obvious mixture of different types of K⁺ channels.

Figure 6. Carbachol (10 μm) inhibited the activity of the M-type K⁺ channels

A, one example recording from the soma. The membrane was held at −66 mV and depolarized to +14 mV for 30 s. Channel activities between 10 and 20 s after the start of the voltage step were analysed. Bath application of 10 μm carbachol reduced channel activity. B, conductance of this channel was estimated to be 9 pS, which is within the range for M-type K⁺ channels. C, carbachol was perfused for 1 min in the bath. Channel activities were inhibited, and the effect could be washed off. D, similar experiments were performed in three recordings, all of which were from the soma. Carbachol inhibited channel activities of the M-type K⁺ channels in all three cases, with a mean of 41% inhibition as compared to control (P < 0.05).

Figure 7. A slowly inactivating component of macroscopic K⁺ currents

The recording was made 170 μm from the soma, in which a double-pulse protocol was used to separate the slowly inactivating component from the non-inactivating component of the macroscopic K⁺ current. In the voltage protocol, two steps to +53 mV (from a holding potential of −97 mV, each lasting 400 ms) were separated by a 5 ms hyperpolarization to −97 mV. Current trace was an average of 5 sweeps. Subtracting the non-inactivating component (b) from the total current (a) resulted in isolation of a slowly inactivating component with τ = 105 ms. (τ = 110 ms with an exponential fitting of the total current (a) before subtraction.)

Figure 8. Single D-type K⁺ channels recorded 240 μm from soma

A, three identical channels were present in this recording. The patch membrane was held at −96 mV. 400 ms depolarizing steps of noted potentials activated three K⁺ channels in a voltage-dependent manner. Bottom trace is the ensemble average of channel activities at +54 mV (average of 9 traces). At this potential, current inactivated with a time constant (τ) of 95 ms. B, amplitude histograms of this recording have four peaks, representing current levels when none of the channels was open, when only one channel was open, when two channels were open at the same time, and when three channels were open at the same time. Distances between the peaks were nearly identical, indicating that the channels were of similar conductance. C, amplitude scatter could be fitted with three lines with slope values of 19 pS (γ), 37 pS (2γ) and 50 pS (3γ), respectively. D, activation and inactivation curves were constructed using peak conductance and peak current values measured from ensemble averages at noted membrane potentials. Activation and inactivation are both fitted with single Boltzmann equations. For activation, V_½ = +4 mV, k = 6.4. For inactivation, V_½ = −20 mV, k = 7.9.

Figure 9. Single dendritic A-type K⁺ channel

A, single, fast inactivating A-type K⁺ channel recorded 120 μm from soma. The patch was held at −95 mV, and voltage steps of noted potentials were given to activate the channel. Bottom trace is ensemble average at +55 mV (average of 28 traces). Current inactivated with τ = 27 ms in this particular recording. B, current amplitude scatter graph was fitted with a line that gave a single-channel conductance of 5 pS for this channel. C, activation (n = 6) and inactivation (n = 3) curves were constructed using peak conductance and peak current values measured from ensemble averages at noted membrane potentials. Activation and inactivation were both fitted with single Boltzmann equations. For activation, V_½ = +5 mV, k = 16.1. For inactivation, V_½ = −64 mV, k = 8.9.

Figure 10. Summary of multiple types of dendritic K⁺ channels

Four types of voltage-dependent K⁺ channels were identified with single-channel analysis.