| Summary: | A novel rat brain endoplasmic reticulum (ER) anion channel was investigated. The channel is co-localised with ryanodine-sensitive Ca<SUP>2+</SUP>-release channels, and physiologically may function as a charge compensator in the ER membrane to regulate Ca<SUP>2+</SUP>-release. Similar channels were reconstituted from sheep brain microsomes and these were partially-purified and reconstituted for macroscopic flux assays. Brain microsomes were incorporated into voltage-clamped planar lipid bilayers. This is a very powerful technique to study single channels from intracellular membranes such as the ER. Channel gating was voltage-dependent and channels opened in bursts with more activity at positive intra-ER (luminal) voltages. The channel displayed multiple closed states including long "interburst" periods where the channel appeared to be inactive. The single-channel slope conductance was 70 pS in symmetrical 250 mM Choline C1 with currents saturating at 170 pS in high concentrations of this salt. Exclusion sizing of ions suggested that the pore had a minimum diameter of approximately 7 Å, and the channel was permeant to both anions and cations with anion selectivities SCN<SUP>-</SUP>> NO<SUB>3</SUB><SUP>-</SUP>> Br<SUP>-</SUP>> Cl<SUP>-</SUP> > F<SUP>-</SUP>. The channel displayed 3 subconductance states with relative amplitudes of 22%, 45% and 70% irrespective of the permeant cation. These results suggest that the channel was functionally behaving as a four-barrelled multimer, and openings to subconductance levels were well-described by the binomial equation. A range of putative C1<SUP>-</SUP> channel blockers were applied. Indanyloxyacetic acid-94 (IAA-94) was studied in detail and found to block the channel from the cytoplasmic side on an intermediate timescale with a K<SUB>i</SUB> of 35 μM at -30mV. Block was voltage-dependent, and relieved by negative luminal voltages. 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) gave an intermediate use-dependent block at 20 μM, and 4,4'-diisothiocyanatostilbene-2,2'- disulphonic acid (DIDS) completely blocked the channel from the cytoplasmic side at 15 μM. NPPB and DIDS showed irreversible inhibition. HEPES added to both chambers at 10mM appeared to block individual protomers on a slow timescale. Single sheep brain ER anion channels showed very similar gating and pharmacological properties to the rat brain channel.
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