Properties of voltage-gated potassium currents in nucleated patches from large layer 5 cortical pyramidal neurons of the rat

Abstract

Voltage-gated potassium currents were studied in nucleated outside-out patches obtained from large layer 5 pyramidal neurons in acute slices of sensorimotor cortex from 13- to 15-day-old Wistar rats (22-25 C). Two main types of current were found, an A-current (IA) and a delayed rectifier current (IK), which were blocked by 4-aminopyridine (5 mM) and tetraethylammonium (30 mM), respectively. Recovery from inactivation was mono-exponential (for IA) or bi-exponential (for IK) and strongly voltage dependent. Both IA and IK could be almost fully inactivated by depolarising prepulses of sufficient duration. Steady-state inactivation curves were well fitted by the Boltzmann equation with half-maximal voltage (V ) and slope factor (k) values of -81.6 mV and -6.7 mV for IA, and -66.6 mV and -9.2 mV for IK. Peak activation curves were described by the Boltzmann equation with V and k values of -18.8 mV and 16.6 mV for IA, and -9.6 mV and 13.2 mV for IK. IA inactivated mono-exponentially during a depolarising test pulse, with a time constant ( approximately 7 ms) that was weakly dependent on membrane potential. IK inactivated bi-exponentially with time constants ( approximately 460 ms, approximately 4.2 s) that were also weakly voltage dependent. The time to peak of both IA and IK depended strongly on membrane potential. The kinetics of IA and IK were described by a Hodgkin-Huxley-style equation of the form mNh, where N was 3 for IA and 1 for IK. These results provide a basis for understanding the role of voltage-gated potassium currents in the firing properties of large layer 5 pyramidal neurons of the rat neocortex.

Figure 7. Prepulse methods for isolating I_A and I_K, and peak current activation plots

A, prepulse method for separating I_A. The membrane potential was alternately stepped to either -117 or -37 mV for 50 ms immediately before stepping to the test potential. The resultant membrane currents (thick trace: prepulse -117 mV; thin trace: prepulse -37 mV) were pair-wise subtracted to isolate I_A (V_test=+63 mV in the example illustrated). Remaining leak and capacitance currents, which were constant for all test potentials, were removed by subtracting from all episodes the pair-wise subtracted episode for V_test=−77 mV, which elicited no potassium current (see text). B, family of I_A currents isolated using this method, shown expanded in the inset. The pulse protocol is shown in A. Each trace is an average of 20 episodes. The external solution also contained 30 mM TEA to minimise I_K. D, example from another patch for which the external solution did not contain TEA. The pulse protocol is the same as in A and B. Each trace is an average of 30 episodes. E, prepulse method for separating I_K. Following a strongly hyperpolarising conditioning pulse (-117 mV for 450 ms) the membrane potential was stepped to -37 mV for 50 ms to inactivate I_A, then stepped to V_test. A family of the resultant leak-subtracted currents is illustrated. Each trace is an average of 20 episodes. The external solution did not contain any blockers apart from TTX. C, averaged, normalised peak activation plots for I_A, obtained in external solution containing 30 mM TEA (•; n= 12), in external solution containing 100 μM Cd²⁺ plus TEA (□; n= 9), or in external solution containing neither (○; n= 8). The prepulse method was used in all experiments. Error bars (±s.e.m.) are mostly smaller than the symbol size. The superimposed curves are Boltzmann functions with the indicated fit parameters. F, averaged, normalised peak activation plots for I_K, obtained in external solution containing 5 mM 4-AP (•; n= 9) or without 4-AP (○; n= 8). Adding external Cd²⁺ had no effect (not illustrated). The superimposed curves are fitted Boltzmann functions with the indicated fit parameters.

Figure 3. I_A inactivates and recovers from inactivation with single-exponential kinetics

A, onset of steady-state inactivation of I_A at -57 mV. Following a 500 ms prepulse to -87 mV to reduce inactivation, membrane potential was stepped to -57 mV for varying durations (ΔT). The amount of activatible I_A was then assayed by a step to +53 mV. ΔT ranged from 0 to 500 ms. B, plot of the amplitudes of I_A from A, after subtracting residual I_K (Methods), versusΔT. Filled circle is a bracket to Δ T= 0 at the end of the sequence. The superimposed curve is a fitted single exponential, time constant 13.2 ms. C, recovery from inactivation of I_A at -97 mV. I_A was inactivated by a 500 ms prepulse to -47 mV, then recovery at -97 mV was monitored. D, plot of the I_A data in C. The superimposed single exponential fit has a time constant of 40.9 ms. Filled circle is a bracket as before. All data in this figure are from the same patch with 30 mM TEA external solution. Each trace is an average of 4 episodes. Leak currents have been subtracted.

Figure 4. I_K inactivates and recovers from inactivation with double-exponential kinetics

Similar experiment to Fig. 3, except that the external solution contained 5 mM 4-AP to block I_A and ΔT ranged from 0 to 45 s. A and B, onset of inactivation at -57 mV, fitted with the sum of two exponentials with time constants 178 ms and 8.86 s. C and D, recovery from inactivation at -97 mV, with fitted time constants 213 ms and 1.91 s. Filled symbols in B and D are bracket measurements made at the end of the sequence. Each trace in A and C is a single episode, all recorded from the same patch. Leak currents were not subtracted.

Figure 2. Instantaneous I–V data reveal that I_K has a more hyperpolarised reversal potential than I_A

A, tail current family for I_K, recorded in 5 mM 4-AP. Following a 100 ms step to +53 mV, the membrane potential was stepped to a level ranging from +53 to -117 mV in 10 mV increments. Each trace is an average of 12 interleaved episodes. Leak currents have been subtracted. B, plot of peak instantaneous I_K, from extrapolated exponential fits to the tail currents (Methods), versus tail potential for this patch. The superimposed curve is a quadratic polynomial. The reversal potential for this patch was -86.4 mV. C, tail current family for I_A, recorded in 30 mM TEA and shown expanded in the inset. The pulse protocol was as in A, except the duration of the prepulse to +53 mV was 1.5 ms. Each trace is an average of 6 interleaved episodes. Leak currents have been subtracted. The slowly rising trace in the inset is the estimated time course of the contaminating I_K at +53 mV in this patch. At 1.5 ms the contamination is about 10 % of I_A. D, plot of peak instantaneous I_Aversus tail potential for this patch. The fitted quadratic polynomial gives a reversal potential of -68.7 mV.

Figure 6. Steady-state inactivation data for I_A and I_K

A, inactivation family for I_A, recorded in 30 mM TEA. The prepulse voltage ranged from -127 to -27 mV in 10 mV increments, and its duration was 500 ms. Each trace is an average of 16 episodes. Leak currents have been subtracted. B, averaged, normalised steady-state inactivation plot for peak I_A, after subtracting the residual I_K (Methods). Data were obtained in control external solution (•; n= 8) or in external solution containing 100 μM Cd²⁺ (□; n= 12). Error bars (±s.e.m.) are mostly smaller than the symbol size. The superimposed curves are Boltzmann functions with the indicated fit parameters. C, inactivation family for I_K, recorded in 5 mM 4-AP. The holding potential was set at -117 to -7 mV in 10 mV increments for times varying from 1 to 30 s prior to the test pulse to +53 mV (see text). Each trace is an average of 8 episodes. Leak currents have been subtracted. D, averaged, normalised steady-state inactivation plot for peak I_K (n= 8). The superimposed curve is the Boltzmann function. Adding external Cd²⁺ had no effect (not illustrated).

Figure 8. I_A kinetics are well fitted by an equation of the form m^Nh

A, voltage dependence of parameters describing the kinetics of I_A. Data are from currents recorded in 30 mM TEA to reduce I_K (•) or in the absence of TEA, using a prepulse method (Fig. 7A) to reduce I_K (○). Aa, time from the foot of I_A to its peak. Ab, inactivation time constant obtained from the fit of a single exponential plus a constant (to account for residual I_K) to the decay phase of I_A. Ac, order (N) obtained from a fit of the equation m^Nh to families of I_A currents, as shown in B and C. Here m incorporates a single exponential rise and h a single exponential decay, and N was allowed to vary. Each point is mean ±s.e.m. (n= 14 for filled circles; n= 4-9 for open circles). The smooth curves superimposed on the filled circles are fits of the TEA data to empirical functions that are summarised in Table 1. The horizontal dashed line in Ac indicates the value of N chosen to calculate the fits summarised in Fig. 10A. B, activation family of I_A (dots) with superimposed fits to the equation m^Nh (smooth curves). Each trace is an average of 8 episodes. Leak currents were subtracted and the external solution contained 30 mM TEA. C, the same family on an expanded time scale.

Figure 10. Parameters for a Hodgkin-Huxley model of I_A and I_K

A, mean τ_m (a) and m_∞³ (b) for I_A, plotted against membrane potential. Filled symbols in a were obtained by fitting eqn (1) (Methods) with N= 3 to plots of I_Aversus time. Open symbols in a were obtained as 3 ×τ_tail, where τ_tail was obtained by fitting a single exponential to the I_A tail currents recorded in instantaneous current-voltage experiments (Fig. 2A). The superimposed smooth curve is described in Table 1. Filled symbols in b were obtained from the above fit of eqn (1), which gave I_max (the extrapolated maximal current in the absence of inactivation). I_max was converted to conductance (Methods) and plotted against membrane potential. The superimposed continuous curve is the Boltzmann function raised to the power 3. The dashed curve is the cube root of the continuous curve and thus represents m_∞; the Boltzmann fit parameters for the dashed curve are given in the figure. Ba and b, the same model parameters for I_K. These were obtained as for I_A, except N= 1, and the factors 3 were replaced by 1. The superimposed smooth curve in a is described in Table 2; that in b is the Boltzmann function with the indicated parameters.

Figure 9. I_K kinetics are well fitted by m^Nh

A, voltage dependence of parameters describing the kinetics of I_K. Data are from currents recorded in 5 mM 4-AP to reduce I_A (•) or in the absence of 4-AP, using a prepulse method (Fig. 7E) to reduce I_A (○). Aa, time to peak, obtained from short timebase leak-subtracted I_K families, like those in B. Ab, decay time constants and the ratio of their amplitudes (Ac) obtained from fits to long timebase I_K families without leak subtraction, like those in C. Each point is mean ±s.e.m. (n= 7). The superimposed smooth curves are fits to empirical functions that are summarised in Table 2. The horizontal dashed line in Ad indicates the value of N chosen to calculate the fits summarised in Fig. 10B. B, activation family of I_K currents (dots) with superimposed fits to the equation m^Nh (smooth curves, mostly obscured by the data). Each trace is an average of 8 episodes. Leak currents were subtracted and the external solution contained 5 mM 4-AP. C, I_K family from a different patch, using a long (20 s) step to the test potential (cf. 500 ms in B). The decay phase was fitted to the sum of two exponentials, plus a constant offset to account for leak current, which was not subtracted in this experiment. Inset shows the currents on an expanded time scale. Each trace is an average of 2 episodes.

Figure 1. Delayed rectifier (I_K) and A-current (I_A) can be pharmacologically distinguished in nucleated patches from large layer 5 cortical pyramidal neurons

Voltage clamp families of potassium currents recorded in three different external solutions (A–C). Following a 500 ms prepulse to -117 mV, the membrane potential (V_m) was stepped to a level ranging from -67 to +73 mV in 20 mV increments (protocol at top). Capacitance transients and linear leak currents have been subtracted using an online subtraction protocol (Methods). A, control external solution, without potassium channel blockers. B, external solution with 5 mM 4-AP. The remaining current is I_K. C, external solution with 30 mM TEA. I_K is reduced, emphasising I_A. All external solutions also contained 0.5 μM TTX. Each trace is an average of 4 (A) or 8 (B and C) episodes. Each patch is from a different neuron, but the current waveform was stereotypical in each solution.

Figure 5. I_A and I_K inactivate and recover from inactivation with voltage-dependent time constants

A, mean time constants (±s.e.m.) for onset of or recovery from inactivation of I_A, plotted against membrane potential, for control external solution (•) and external solution containing 100 μM Cd²⁺ (□). Note the logarithmic ordinate. A single time constant described the inactivation onset/recovery of I_A at each membrane potential. Ba, a similar plot for I_K. Two time constants were needed to describe the inactivation onset/recovery of I_K, except at V_recov=−117 mV where a single exponential sufficed (○). External Cd²⁺ had no effect on I_K. Bb, mean ratio Amp_fast/Amp_slow for the onset/recovery of I_K, where Amp_fast is the amplitude of the fitted fast component and Amp_slow is that of the slow component. The continuous lines in all panels are fits to empirical functions that are summarised in Tables 1 and 2.