Proceedings of The Physiological Society

Durham University (2010) Proc Physiol Soc 21, C15 and PC15

Oral Communications

In lanceolate endings of rat hair follicles the small conductance Ca2+-activated K+ channel SK3 is found mainly in glial cells

F. C. Shenton1, R. W. Banks1, G. S. Bewick2

1. School of Biological & Biomedical Sciences, Durham University, Durham, United Kingdom. 2. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.

Autogenic modulation of mechanoreceptor excitability by Ca2+-dependent glutamate release from synaptic-like vesicles (SLVs) has been shown using the muscle spindle as a model (1). The excitability of lanceolate endings of hair follicles may be similarly regulated (2). In spindles Ca2+ entry through P/Q type channels activates KCa channels (BK or SK) to regulate afferent firing (3). In excitatory synapses of mouse hippocampus SK3 is a presynaptic channel (4) where it is probably involved in regulating neurotransmitter release. SK3 might therefore play a role in modulating glutamate release from SLVs. We have now studied SK3 expression in spindles and lanceolate endings, plus their associated satellite glial cells (SLGs) by immunocytochemistry. Synaptophysin (SYN, a marker of SLVs) was used to label sensory endings while the Ca2+-binding protein, S100 was used to identify SLGs. Adult rats (2) were deeply anaesthetized with sodium pentobarbitone (45 mg kg−1, I.P.) and fixed by transcardial perfusion (4% (w/v) paraformaldehyde in 0.1M phosphate buffer, pH 7.4; all procedures in accordance with ASPA 1986). Immunofluorescence labelling was carried out on 10µm cryosections of pinna skin. Sections were double stained with one of four antibody combinations: 1) anti-SK3 (5µg/ml, goat polyclonal Santa Cruz Biotechnology) + anti-SYN (1μg/ml, mouse monoclonal Millipore); 2) anti-SK3 + anti-S100 (1:400, mouse monoclonal Santa Cruz Biotechnology); 3) anti-ASIC2 (5µg/ml, goat polyclonal Santa Cruz Biotechnology) + anti-SYN; 4) anti-ASIC2 + anti-S100. Secondary antibodies were Alexa Fluor (AF) conjugated (AF 594 goat anti-rabbit and AF 488 goat anti-mouse, Invitrogen). Images were taken with a Leica SP5 Laser Scanning Microscope. Colocalisation of SK3 and ASIC2 reactivity with either S100 or SYN labelling was assessed by Pearson’s correlation coefficient (r) using LAS AF Lite software (Leica Microsystems CMS GmbH). We were unable to demonstrate SK3 in spindle terminals but it was present in the SLGs of lanceolate endings and, at lower levels, in the terminals themselves. Correlation of SK3 with S100 (r = 0.34 + 0.05, mean + SE) was greater than ASIC2 with S100 (0.15 + 0.03), P < 0.01. SK3 correlation with SYN (0.24 + 0.02) was less than that of ASIC2 with SYN (0.42 + 0.03), P < 0.01. SK3 correlation with S100 was greater than with SYN, but this did not reach statistical significance. Thus, SK3 was expressed predominantly in SLGs. By contrast immunolabelling for the Na+ ion channel ASIC2, previously reported in lanceolate endings (5) and SYN were largely coincident. Our data suggest that in lanceolate endings SK3 is expressed predominantly in SLG’s. SK3 channels could play a role in shaping SLG responses to fluctuations in intracellular Ca2+ and thereby indirectly influence afferent excitability of the terminals.

Where applicable, experiments conform with Society ethical requirements