Abstract
Kv3.1 and Kv3.3 subunits are highly expressed in the auditory brainstem, with little or no mRNA for Kv3.2 or Kv3.4. Changes in Kv3 currents and action potential (AP) firing were analysed from wild-type, Kv3.1 and Kv3.3 knockout (KO) mice. Both Kv3.1 and Kv3.3 immunostaining was present and western blots confirmed loss of subunit protein in the respective KO. Medial nucleus of the trapezoid body (MNTB) AP repolarization utilized Kv3.1 and/or Kv3.3; while in the lateral superior olive (LSO) Kv3.3 was essential. Voltage-gated calcium currents were unchanged between the genotypes. But APs evoked higher [Ca2+ ]i in LSO than MNTB neurons; and were highest in the Kv3.3KO, consistent with longer AP durations. High frequency stimulation increased AP failure rates and AP latency in LSO neurons from the Kv3.3KO, underlining the physiological consequences for binaural integration. LSO neurons require Kv3.3 for functional Kv3 channels, while MNTB neurons can utilize either Kv3.1 or Kv3.3 subunits. Kv3 voltage-gated potassium channels mediate action potential (AP) repolarization. The relative importance of Kv3.1 and Kv3.3 subunits for assembly of functional channels in neurons of the auditory brainstem was examined from the physiological perspective that speed and precision of AP firing are crucial for sound source localization. High levels of Kv3.1 and Kv3.3 mRNA and protein were measured, with no evidence of compensation by Kv3.2 or Kv3.4 in the respective knockout (KO) mouse. Using the KOs, composition of Kv3 channels was constrained to either Kv3.1 or Kv3.3 subunits in principal neurons of the medial nucleus of the trapezoid body (MNTB) and lateral superior olive (LSO); while TEA (1mm) was employed to block Kv3-mediated outward potassium currents in voltage- and current clamp experiments. MNTB neuron APs (half-width 0.31±0.08ms, n=25) were fast, reliable, and showed no distinction between channels assembled from Kv3.1 or Kv3.3 subunits (in the respective KO). LSO AP half-widths were also fast, but absolutely required Kv3.3 subunits for fast repolarization (half-widths: 0.25±0.08ms, n=19 wild-type, 0.60±0.17ms, n=21 Kv3.3KO, p=0.0001). The longer AP duration increased LSO calcium influx and AP failure rates, and increased AP latency and jitter during high frequency repetitive firing. Both Kv3.1 and Kv3.3 subunits contribute to Kv3 channels in the MNTB (and compensate for each other in each KO); in contrast, LSO neurons require Kv3.3 subunits for fast repolarization and to sustain AP firing during high frequency stimulation. In conclusion, Kv3 channels exhibit both redundancy and Kv3.3 dominance between the brainstem nuclei involved in sound localization.
Highlights
Voltage-gated potassium channels are key regulators of neuronal excitability, action potential (AP) threshold, repolarization and waveform
Whole-cell patch-clamp recordings were made from medial nucleus of the trapezoid body (MNTB) or lateral superior olive (LSO) principal neurons in an in vitro brain slice preparation prepared from CBA/CaCrl mice aged P14–P25
Superimposed current traces from one MNTB and one LSO principal neuron are shown in Fig. 1A and B, with the current–voltage (I/V) relationship plotted for peak and sustained currents in Fig. 1C and D, respectively
Summary
Voltage-gated potassium channels are key regulators of neuronal excitability, action potential (AP) threshold, repolarization and waveform. Homomeric Kv3 channels have been well characterized by expression in recombinant systems (Grissmer et al 1994; Macica & Kaczmarek, 2001; Desai et al 2008), but in many brain regions multiple Kv3 subunit genes are expressed in the same neurons (Perney et al 1992; Weiser et al 1994; 1995; Chang et al 2007), including neurons in the auditory brainstem This has been taken as evidence of functional redundancy, since early studies of Kv3.1 knockouts (KOs) showed only a mild behavioural phenotype (with changes in cortical gamma and delta oscillations, Joho et al 1999), while Kv3.3KOs showed no obvious behavioural phenotype (Espinosa et al 2001). The auditory brainstem and the medial nucleus of the trapezoid body (MNTB) in particular, is a well-established model for studies of Kv3 channel physiology in the context of auditory processing This investigation exploited KO mice to generate Kv3 channels of reduced complexity in this native physiological system, allowing the study of homomeric Kv3 currents in neurons which normally express multiple subunits. In the MNTB, fast spiking was maintained in both Kv3.1 and Kv3.3 KO mice, implying that either subunit compensated for the absence of the other
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