Immunolocalization of the Ca²⁺‐activated K⁺ channel Slo1 in axons and nerve terminals of mammalian brain and cultured neurons

Abstract

Ca(2+)-activated voltage-dependent K(+) channels (Slo1, KCa1.1, Maxi-K, or BK channel) play a crucial role in controlling neuronal signaling by coupling channel activity to both membrane depolarization and intracellular Ca(2+) signaling. In mammalian brain, immunolabeling experiments have shown staining for Slo1 channels predominantly localized to axons and presynaptic terminals of neurons. We have developed anti-Slo1 mouse monoclonal antibodies that have been extensively characterized for specificity of staining against recombinant Slo1 in heterologous cells, and native Slo1 in mammalian brain, and definitively by the lack of detectable immunoreactivity against brain samples from Slo1 knockout mice. Here we provide precise immunolocalization of Slo1 in rat brain with one of these monoclonal antibodies and show that Slo1 is accumulated in axons and synaptic terminal zones associated with glutamatergic synapses in hippocampus and GABAergic synapses in cerebellum. By using cultured hippocampal pyramidal neurons as a model system, we show that heterologously expressed Slo1 is initially targeted to the axonal surface membrane, and with further development in culture, become localized in presynaptic terminals. These studies provide new insights into the polarized localization of Slo1 channels in mammalian central neurons and provide further evidence for a key role in regulating neurotransmitter release in glutamatergic and GABAergic terminals.

Down-regulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

“…In general, granule cells and pyramidal cells are devoid of somatic staining. Similar distribution of BK channel staining was previously reported in the mouse hippocampus using either monoclonal anti-BK 690-1196 (Misonou et al, 2006) or polyclonal anti-BK antibodies (Sailer et al, 2006).…”

“…The abundance of BK channels on such subcellular locations is differentially regulated across several neuronal phenotypes. In the hippocampus, for instance, BK channels are highly expressed at the Schaffer's collateral pathway and mossy fibers of granule cells (Hu et al, 2001;Knaus et al, 1996;Misonou et al, 2006;Sailer et al, 2006).…”

Profiling the Phospho-status of the BKCa Channel α Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

“…The C-DEC splice variant generates an extended C terminus resulting in BK␣ with enhanced retention of newly synthesized BK Ca channels in the endoplasmic reticulum, which finally results in decreased cell surface expression (60,61). BK Ca channels are robustly expressed in mammalian central neurons (62,63) and are predominantly localized to axons and presynaptic terminals (17,37) but are also present in certain neuronal somata and dendrites (18,64). In rat brain, BK␣ splice variants may be differentially expressed in distinct cell types or different compartments of the same neuron.…”

“…One set employed monoclonal antibody-based immunopurification from rat brain membranes prepared from freshly isolated adult whole rat brains as described (35) and solubilized by 1% Triton X-100 or 1% dodecyl-maltoside. BK␣ was affinity-purified using the monoclonal antibody L6/60 (termed anti-BK␣_1) (36,37) immobilized on protein G agarose beads. L6/60 binds within amino acid residues 729 -930 of mouse BK␣ (UniProt/Swiss Prot accession number Q08460).…”

Presynaptic Ca2+/Calmodulin-Dependent Protein Kinase II Modulates Neurotransmitter Release by Activating BK Channels atCaenorhabditis elegansNeuromuscular Junction

“…The BK channel is an attractive candidate. In virtually all synapses examined, the BK channel is a prominent K ϩ channel at presynaptic nerve endings (Anderson et al, 1988;Farley and Rudy, 1988;Lindgren and Moore, 1989;Tabti et al, 1989;Morita and Barrett, 1990;Sivaramakrishnan et al, 1991;Robitaille et al, 1993;Wangemann and Takeuchi, 1993;Katz et al, 1995;Vatanpour and Harvey, 1995;Knaus et al, 1996;Sun et al, 1999;Zhou et al, 1999;Yazejian et al, 2000;Misonou et al, 2006), where it colocalizes with voltage-gated Ca 2ϩ channels (Robitaille et al, 1993;Issa and Hudspeth, 1994;Yazejian et al, 1997;Yazejian et al, 2000), and may serve as an important negative regulator of neurotransmitter release (Robitaille et al, 1993;Wang et al, 2001;Raffaelli et al, 2004). Several lines of evidence suggest that BK channel activity may be modulated by CaMKII: (1) BK channels reconstituted into artificial lipid bilayers are activated by ATP, which is blocked by a CaMKII inhibitor (Muller et al, 1996); (2) interactions between 14-3-3 and Slob, which are proteins that control Drosophila BK channel activity, may be modulated by CaMKII (Zhou et al, 1999); and (3) BK channels contribute to afterhyperpolarization of mouse vestibular nucleus neurons in a CaMKII-dependent manner (Nelson et al, 2005).…”