Coassembly of Big Conductance Ca2+-activated K+ Channels and L-type Voltage-gated Ca2+ Channels in Rat Brain

Morten Grunnet,Walter A Kaufmann

doi:10.1074/jbc.m402254200

Abstract

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.

Highlights

Based on electrophysiological studies, Ca2؉-activated K؉ channels and voltage-gated Ca2؉ channels appear to be located in close proximity in neurons
Immunohistochemistry was performed on serial, sagittal cut rat brain sections. These studies were undertaken to obtain an overview on the localization of BK channels as well as ␣1C (CNC) and ␣1D (CND) L-type Ca2ϩ channels rather than a detailed expression profile of each channel type at the cellular level
The present study was carried out to investigate a possible interaction between Ca2ϩ-activated Kϩ channels and voltagegated Ca2ϩ channels in rat brain

Summary

EXPERIMENTAL PROCEDURES

Materials—An iberiotoxin double mutant was engineered, purified, and radioiodinated with 125I (125I-IbTX-D19Y/Y36F) as described previously [29]. Membranes were washed in washing buffer consisting of 0.5% (w/v) Triton X-100, 0.1% (w/v) Tween 20, dissolved in 150 mM NaCl, 20 mM Tris/HCl, pH 7.4, and incubated with affinity-purified alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies (Sigma) diluted 1:2,000 in blocking buffer for 90 min on a rocking plate at room temperature. Rat brain membranes were solubilized before loading at the column This was obtained by washing 2-ml membranes in 20 ml of buffer consisting of 10 mM NaCl, 20 mM Tris, 0.1% BSA followed by a 10-min centrifugation at 48,000 ϫ g. Membranes were centrifuged for 30 min at 106.000 ϫ g and the supernatant containing solubilized membranes loaded at the column in a total volume of 7 ml of 2% (w/v) digitonin, 150 mM NaCl, 20 mM Tris/HCl, pH 7.4 and incubated overnight at 4 °C under gentle rotation. Protein Determination—The protein concentrations of the membrane preparations were determined according to Bradford using BSA as a standard

RESULTS

DISCUSSION

Channels Brain and