Anionic selectivity sequence of the Cl(-)-H+ symporter in the synaptosomal preparation from rat brain cortex

Cytosolic pH regulation in synaptosomes remains poorly understood. While the Na+/H+ exchanger is a confirmed H+ transport mechanism, alternative pathways are suspected. Prior studies suggest a Cl−-H+ symporter contributes to pH regulation. This paper investigates anion selectivity of that proposed transport mechanism. The stability of Fura-2 ratios in different anion solutions is a novel approach. No prior work had resolved the specific anion preferences of this symporter. The role of anions in membrane depolarization is a key uncertainty. This gap motivated a detailed analysis of anion effects on cytosolic pH recovery. The study builds on established knowledge of H+ transporters in neural membranes.

The study aimed to characterize anion selectivity of a proposed Cl−-H+ symporter in synaptosomes. The specific problem is understanding how different anions influence cytosolic pH recovery. The motivation stems from gaps in pH regulation mechanisms beyond the Na+/H+ exchanger. The focus is on anion-specific effects on membrane depolarization. The goal is to identify which anions promote or inhibit pH recovery. This addresses a technical question about transport specificity. The study design allows measuring Fura-2 ratios in various anion solutions. The approach isolates the symporter's role from other transporters.

The study used synaptosomal preparations from rat brain cortex. Fura-2 fluorescence ratios were measured to track cytosolic pH changes. Solutions with different anions were tested for pH recovery effects. Gluconate and sulfate solutions were compared to 50 mM K solution. Anionic selectivity was determined by recovery rates from alkalinization. PCMB inhibition was tested at 10 µM concentration. Niflumic acid, 9AC, Bumetanide, and CCCP were used as inhibitors. The experimental setup allowed isolating the symporter's activity from other transporters.

The Fura-2 ratio remained stable in gluconate and sulfate solutions. This stability suggests no plasma membrane depolarization occurred. Anionic selectivity sequence was NO3− > Br− > Cl− >> I− = isethionate = sulfate = methanesulfonate = gluconate. PCMB inhibited gluconate-dependent alkalinization by 30 ± 6%. Niflumic acid, 9AC, Bumetanide, and CCCP had no inhibitory effect. The recovery rate directly correlates with anion type. The symporter shows highest activity with nitrate and bromide. The lowest activity was observed with iodide and isethionate.

The study confirms a Cl−-H+ symporter's role in pH regulation. Anion selectivity sequence was directly observed through recovery rates. PCMB partially inhibits gluconate-dependent alkalinization. Specific inhibitors like Niflumic acid and Bumetanide had no effect. The symporter's activity is independent of these transporters. The findings support the symporter's acidifying mode function. The results align with the authors' prior hypothesis about this mechanism. The study provides direct evidence for anion-specific transport activity.