Proceedings of The Physiological Society
University of Cambridge (2004) J Physiol 555P, C32
Evidence that the Kv3.1b subunit isoform contributes to action potential repolarisation in neurones within the nucleus of the solitary tract in rat
Mark L. Dallas, Susan A. Deuchars, David I. Lewis and Jim Deuchars
School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK
The voltage gated potassium channels Kv3 subfamily has been specifically implicated in the fast spiking neuronal phenotype, facilitating brief action potentials (Rudy et al. 1999). We have localised Kv3.1b immunoreactivity within specific regions of the nucleus of the solitary tract (NTS, Deuchars & Atkinson, 2001) and shown neurones within these regions to be sensitive to 4-AP and TEA (Dallas et al. 2002). Since no pharmacological tools are available to distinguish between the Kv3.1 isoforms we sought to determine the specific role of a Kv3.1b subunit, using a novel approach of intracellular application of a Kv3.1b specific antibody.
Male Wistar rats (15-21days) were humanely killed by anaesthetising with sodium pentobarbitone (120 mg kg-1, I.P.) followed by transcardial perfusion with sucrose aCSF and subsequent decapitation. Whole cell patch clamp recordings were made from neurones within the NTS (n = 7) and the dorsal vagal nucleus (DVN, n = 7) at room temperature. The primary antibody raised against a Kv3.1b channel subunit (1:1000, Alomone Labs) was added to the intracellular solution contained within the patch pipette. In NTS neurones this resulted in progressive prolongation of the action potential duration (from 4.6 ± 0.7 ms to 7.7 ± 0.8 ms at 30 min, mean ± S.E.M.; P < 0.05, Student's paired t test). After 30 min 4-AP (30µM) produced a further significant increase in the AP duration (7.7 ± 0.8 ms to 10.1 ± 1.1, P < 0.05), which was reversed upon washout of the 4-AP. Repeated applications of 4-AP revealed a progressive blockade due to the antibody since at 65 min 4-AP had no further effect on the action potential duration (11.2 ± 0.8 ms to 11.4 ± 0.7 ms, P > 0.05). In the DVN neurones, application of the antibody to the pipette did not lead to a significant increase in the AP duration (5.8 ± 0.6 ms to 5.6 ± 0.3 ms, P > 0.05). After 30 and 65 mins application of 4-AP and TEA did not significantly alter the electrophysiological characteristics of the neurones (4-AP; 5.6 ± 0.3 ms to 5.7 ± 0.8 ms, TEA; 5.75 ± 0.6 to 5.5 ± 0.9 ms, P > 0.05).
These data suggest that the introduction of a Kv3.1b antibody into the neurone specifically disrupts the channel subunit and that this subunit has a role in the action potential repolarisation in NTS neurones.
The University of Leeds and the Wellcome Trust supported this work
Where applicable, experiments conform with Society ethical requirements