Localization of the Slack potassium channel in the rat central nervous system

Abstract

The Slack gene encodes a voltage-dependent K(+) channel that has a unitary conductance of approximately 60 pS. Evidence from heterologous expression studies suggests that Slack channel subunits can also combine with the Slo subunit to generate Ca(2+)-activated K(+) channels of larger conductances. Nonetheless, the function of Slack in the brain remains to be identified. We have now generated an affinity-purified antibody against the N-terminal of rat Slack, for biochemical and immunohistochemical studies. The antibody recognized Slack in transiently transfected CHO cells both by immunocytochemistry and by Western blot analysis. The antibody also detected a single band in rat brain membranes. The localization of Slack in rat brain slices was then determined using the antibody. Most prominent Slack immunoreactivity occurs in the brainstem, in particular the trigeminal system and reticular formation, where very intense staining was found in both cell bodies and axonal fibers of associated nuclei. Labeling was also very strong in the vestibular and oculomotor nuclei. Within the auditory system, the medial nucleus of the trapezoid had a robust signal consistent with staining of the giant presynaptic terminals. Strong Slack immunoreactivity was present in the olfactory bulb, red nucleus, and deep cerebellar nuclei. There was labeling also in the thalamus, substantia nigra, and amygdala. The only cortical region in which Slack immunoreactivity was detected was the frontal cortex. The subcellular and regional distribution of Slack differs from that previously reported for the Slo channel subunit and suggests that Slack may also have an autonomous role in regulating the firing properties of neurons.

NAD+Activates KNaChannels in Dorsal Root Ganglion Neurons

“…Using a previously characterized antibody against Slack channels (Bhattacharjee et al, 2002), we found Slack immunoreactivity in all types of rat DRG neurons (Fig. 1).…”

“…Then sections were incubated with a mixture of primary antibodies in PBS containing 5% BSA overnight at 4°C. Primary antibodies included mouse antineurofilament antibody (1:400; Millipore Bioscience Research Reagents), calcitonin gene-related peptide (CGRP) antibody (1:2500; Sigma), and chicken anti-Slack antibody (1:1000) (Bhattacharjee et al, 2002). After several rinses, secondary antibodies Alexa Fluor 633 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, and Alexa Fluor 546 goat anti-chicken were added (1:1000) for 2 h. Coverslips were then mounted on slides using Prolong Gold antifade reagent with 4Ј,6Ј-diamidino-2-phenylindole dihydrochloride.…”

PKA-Induced Internalization of Slack KNaChannels Produces Dorsal Root Ganglion Neuron Hyperexcitability

“…Neurons were permeabilized with 0.1% Triton X-100 for 4 min and then washed again in PBS. Neurons were incubated with an anti-Slack antibody (1:1000) (Bhattacharjee et al, 2002) at 4°C overnight followed by a secondary AlexaFluor 546 goat anti-chicken antibody (1:1000) for 2 h at room temperature. After three washes in PBS, coverslips were then mounted on slides and imaged.…”

Loss of Kv3.1 Tonotopicity and Alterations in cAMP Response Element-Binding Protein Signaling in Central Auditory Neurons of Hearing Impaired Mice

“…All groups of mice reacted strongly to a loud acoustic stimulus with the exception of the 8-month-old C57BL/6 and the 8-month-old DBA/2J animals, which showed little or no response. Immunohistochemistry was performed as described previously (Bhattacharjee et al, 2002). Anti-CREB and anti-pCREB were purchased from Cell Signaling Technology (Beverly, MA).…”