Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (ICa) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of ICa and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca2+ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α2δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region

G protein-coupled receptor-mediated calcium signaling in astrocytes

Astrocytes express a large variety of G~protein-coupled receptors (GPCRs) which mediate the transduction of extracellular signals into intracellular calcium responses. This transduction is provided by a complex network of biochemical reactions which mobilizes a wealth of possible calcium-mobilizing second messenger molecules. Inositol 1,4,5-trisphosphate is probably the best known of these molecules whose enzymes for its production and degradation are nonetheless calcium-dependent. We present a biophysical modeling approach based on the assumption of Michaelis-Menten enzyme kinetics, to effectively describe GPCR-mediated astrocytic calcium signals. Our model is then used to study different mechanisms at play in stimulus encoding by shape and frequency of calcium oscillations in astrocytes.Comment: 35 pages, 6 figures, 1 table, 3 appendices (book chapter

Ca2+ signaling in astrocytes from Ip3r2−/− mice in brain slices and during startle responses in vivo

Intracellular Ca(2+) signaling is considered important for multiple astrocyte functions in neural circuits. However, mice devoid of inositol triphosphate type 2 receptors (IP3R2) reportedly lack all astrocyte Ca(2+) signaling, but display no neuronal or neurovascular deficits, implying that astrocyte Ca(2+) fluctuations play no role(s) in these functions. An assumption has been that loss of somatic Ca(2+) fluctuations also reflects similar loss within astrocyte processes. Here, we tested this assumption and found diverse types of Ca(2+) fluctuations within astrocytes, with most occurring within processes rather than in somata. These fluctuations were preserved in IP3R2(−)(/)(−) mice in brain slices and in vivo, occurred in endfeet, were increased by G-protein coupled receptor activation and by startle-induced neuromodulatory responses. Our data reveal novel Ca(2+) fluctuations within astrocytes and highlight limitations of studies that used IP3R2(−)(/)(−) mice to evaluate astrocyte contributions to neural circuit function and mouse behavior