Single-channel properties of a rat brain endoplasmic reticulum anion channel

Abstract

Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.

Modulation of the hepatocyte rough endoplasmic reticulum single chloride channel by nucleotide–Mg2+ interaction

“…Our observed chloride channels had a conductance higher than some of the reported chloride channels from cardiac sarcoplasmic reticulum [18] and rat brain endoplasmic reticulum anion channels [5]. VDACs in the mitochondrial outer membrane exhibit a large conductance, 0.45-0.58 nS with a bell-shaped current-voltage relationship in 0.1 M KCl [4,6,33] and thus are incompatible with these results.…”

“…It was inhibited by several compounds including 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), Mg 2+ , and ATP [7], whereas Bégault and Edelman reported an ATP-activated Cl − channel using reconstituted endoplasmic reticulum-enriched pancreatic microsomes [3]. Accordingly, Clark et al found anion channels with different conductances and gating behaviors in rat brain endoplasmic reticulum [5].…”

Fatty Acid-mediated Calcium Sequestration within Intracellular Calcium Pools

“…The effect of DIDS was to block any action of GTP and palmitate in promoting Ca 2ϩ uptake. This action of DIDS was half-maximal between 10 and 30 M in preventing both palmitate-and oxalatedependent Ca 2ϩ accumulation in the presence of GTP (data not shown), clearly within the sensitivity range described above for anion channels of the SR and ER (51,52). DIDS had little inhibitory effect on the initial rate of ATP-dependent Ca 2ϩ pumping in the absence of palmitate or oxalate; at longer time intervals, a slight reduction in Ca 2ϩ accumulation may reflect the permissive role of anion channels in mediating charge equilibration and allowing an increase in the equilibrium level of Ca 2ϩ to be attained within the ER.…”

“…Anion channels in both the SR (45, 47-49) and ER (44, 50) have also been well described. In rabbit SR, there appear to be two different anion channels that are blocked by 8 and 80 M DIDS, respectively (51), and in rat brain ER, anion channels are blocked by DIDS in the 15-100 M range (52). We therefore investigated whether DIDS would modify the action of either palmitate or oxalate in promoting Ca 2ϩ accumulation in the presence of GTP.…”

Rat Brain p64H1, Expression of a New Member of the p64 Chloride Channel Protein Family in Endoplasmic Reticulum

“…Most recordings are from non-neural tissues (3,5,7), and many of these channels may be protein translocation pores rather than conventional ion channels. Anion channels reconstituted from rat brain microsomes (2,45) and cardiac mitoplast membranes (11) are poorly selective for anions versus cations, like p64H1-related channels, but they appear to display different conductances and have different single-channel gating and substate behavior.…”