Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

Abstract

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K(+) current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of approximately 37 nS; it is half-activated at -9.2 +/- 2.1 mV and half-inactivated at -35.9 +/- 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 microm) and high concentrations of extracellular tetraethylammonium (TEA; IC(50) = 11.8 mM), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein approximately 110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin-Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.

Figure 2. Kinetics of the slow high voltage-activated current

A, in the presence of TEA (3 mm) and DTx-I (10 nm) an initial depolarization from −97 to −37 mV activates, and then inactivates the A-current. After 20 ms further voltage steps were applied to activate the remaining voltage-gated current. B, the voltage dependence of activation is fitted by a single exponential; the mean time constant is plotted against voltage and shows an e-fold increase with 17.7 mV depolarization (n = 6) grey trace. C, enlargement of the dash-boxed area in A; an activation curve was measured from these tail currents, measured at the latency indicated by the filled diamond. D, a sustained conditioning voltage step (8 s) over voltages ranging from −107 to +43 mV showed the isolated delayed rectifier to have slow inactivation, which was assessed by measuring the current on stepping to +43 mV (filled triangle). E, a Boltzmann function was fitted to the magnitude of activation tail currents (filled diamonds) and another Boltzmann function was fitted to the inactivation data (filled triangles) from the data shown in C and D (see text for mean data). F, deactivation rates were measured after maximally activating the current by stepping to +53 mV for 15 ms and then stepping to the indicated voltages. Single exponentials fitted the tail currents, with the averaged data plotted in G. Mean data are plotted for 6 neurons. Deactivation accelerated e-fold with 30.6 mV hyperpolarization (grey trace, n = 6; NB, fits close to zero current were omitted as being too small for reasonable accuracy).

Figure 5. A Hodgkin–Huxley model of an MNTB neuron shows that Kv2 regulates the interspike potential and affects availability of Nav channels during high-frequency firing

A, time constants used to generate the model Kv2.2 were obtained from a fit (dashed line) to the activation and deactivation time constants obtained from voltage-clamp experiments (see Fig. 2). B, the magnitudes of the ionic conductances present in the implementation of the single compartment MNTB model adapted from Macica et al. (2003). C, the membrane potential of the model MNTB neuron in response to synaptic trains at 50 Hz (left) and 200 Hz (right) in the presence (black) and absence (grey) of the Kv2.2 conductance; arrows indicate AP peak and interspike potential at the end of each train, respectively. D, the magnitude of the Kv2.2 current passing during the APs in the traces shown above in C. Note that the Kv2.2 current dramatically increases in magnitude at the start of the 200 Hz train (right) but is smaller and relatively stable in the 50 Hz train (left). Note the difference in interspike potential (black arrow) and action potential height (grey arrow).

Figure 1. A slow delayed rectifier in MNTB neurons

A, control outward K⁺ currents evoked by step commands from a pre-pulse voltage of −97 mV to the indicated voltages, in 10 mV increments under voltage-clamp conditions. B, repeat of the same voltage-clamp protocol in the same neuron following perfusion of DTx-I (10 nm) and TEA (3 mm). Arrow indicates unmasking of a small A-type current. C, the mean current–voltage (I–V) relationship for control (black, n = 18) and following DTx and TEA perfusion (grey, n = 18). Note that more than half of the outward current is insensitive to DTx and TEA; this current is therefore not mediated by Kv1 or Kv3 channels.

Figure 3. There were no specific blockers of this slow delayed rectifier

A, in the presence of TEA and DTx-I (black trace) quinine (100 μm) blocked the remaining sustained voltage-gated current (grey trace). The GHK current equation fits the quinine-insensitive current showing that it is not voltage gated, but reflects the K⁺ leak currents. Inset traces show the respective control (black) and test (grey) traces from the same cell (without inactivation of the A-current; pre-pulse potential −97 mV). B, 4-aminopyridine (4-AP, 5–10 mm, n = 4) gives a partial block of the current; inset shows control and test (grey) current traces from the same cell (pre-pulse potential −77 mV). The 4-AP-insensitive current is not fitted by the GHK current equation, indicating that voltage-gated current remains. C, high concentrations of TEA blocked the slow-delayed rectifier. A dose–response curve to TEA was conducted and data pooled from multiple cells. Each point is the mean current from 3–4 neurons. The grey curve is a fit to the Hill equation and gives an IC₅₀ of 11.8 mm. The open triangle was a free parameter to allow for block by 1 mm TEA which was present in all solutions to block Kv3 currents (see Methods). Inset: representative traces are shown from one cell at +13 mV.

Figure 4. Kv2.2 mRNA and protein are present in MNTB principal neurons

A, QRT-PCR of Kv3.1, 2.1 and 2.2 mRNA, expressed relative to Kv3.1. Each value is the mean of 3 separate reactions normalized to actin transcripts. B, Western blots with the Kv2.2 antibody in brain tissue from CBA mice (WT, centre right) and following pre-incubation with the blocking peptide (BP) shows a band at around 110 kDa. The left blots using the same Kv2.2 antibody in brain tissue obtained from homozygous Kv2.2 knockout mice (Lexicon, TF1551) and control littermates (WT, centre left). C, Kv2.2 immunoreactivity (green) is present in the MNTB and VNTB of the superior olivary complex and at lower levels across the auditory brainstem. Bright ‘sparks’ of fluorescence can be seen in the MNTB. D, at higher magnification MNTB principal neurons and the fluorescent sparks are clear. A plot of intensity along the dashed white line shows the incidence of the highly fluorescent sparks (red dashed lines) often alongside a neuronal profile (*). Inset (top) shows blocking peptide negative control. E, a confocal projection of one MNTB neuron double-labelled for Kv2.2 and DAPI (blue nucleus) showing a highly stained putative initial segment region (arrow). F, a confocal projection of a triple-labelled MNTB neuron showing co-localization of Kv2.2 (green) with Kv1.2 (red) in adjacent parts of the axon initial segment (AIS) and DAPI (blue).

Figure 6. Frequency-dependent hyperpolarization of the interspike potential under physiological conditions

A, example 50 Hz (black) and 200 Hz (grey) trains of APs in the same MNTB neuron evoked with identical current trains at the two frequencies. Note the different time scales. Dashed lines indicate the peak of the 1st interspike potential and arrows show the peak of the last. Note that AP amplitudes remain fairly constant throughout, but the hyperpolarization of the interspike interval is greater with higher frequency stimulation. The 1st and 60th action potentials from 50 and 200 Hz overlain (black and grey, respectively) show that only the interspike voltage changes. C, plot of the peak interspike potential for the cell in A.

Figure 7. Sensitivity of I_Na to the interspike potential

A, the steady-state inactivation of I_Na was determined by plotting the peak current measured at −4 mV against the voltage of the 500 ms pre-pulse, which was fitted with a Boltzmann distribution giving a V_½in of −55.4 ± 1.7 mV and a k_in of 6.3 ± 0.1 (n = 5); inset shows example traces. B, the protocol used to asses the sensitivity of I_Na to changes in interspike potential (V_is); a 500 ms −94 mV pre-pulse (to remove all steady-state inactivation) was followed by test pulses to −4 mV interleaved with 5 ms intervals, simulating the time between spikes in a 200 Hz train, as indicated by the overlaid AP (red). C, example traces (all from one cell) generated from the protocol in B, show the sensitivity of I_Na to V_is. Black, V_is hyperpolarized from −55 to −59 mV (identical to Fig. 5D). Blue, V_is is kept constant at −55 mV. Green, V_is depolarized from −55 to −51 mV, mimicking mild summation of the interspike potential. D, average I_Na amplitudes during the trains shown in C; data normalized to 2nd spike to show change from the initial level of inactivation. Asterisks indicate onset of statistical significance.

Figure 8. Age and tonotopic gradients of Kv2.2 current

A, the current measured 40 ms into a +13 mV step depolarization (peak activation) is plotted against postnatal age. A significant increase in the current magnitude is observed with development (statistical significance assessed by ANOVA, P < 0.001). B, the MNTB nucleus was divided into 3 parts, medial (M), intermediate (I) and lateral (L). C, the current measured 40 ms into a +13 mV step depolarization is plotted against location for P12 animals. A significant trend to larger currents in lateral neurons was seen across the tonotopic axis. NB, all data recorded in the presence of 10 nm DTx and 3 mm TEA.