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

Abstract

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.

Protonophore properties of hyperforin are essential for its pharmacological activity

“…4c ), proofing that a decrease, as seen for hyperforin, represents an intracellular acidification. The relatively small negative membrane potential of HEK cells (−50 mV 34 ) represents a limited driving force for protons into the cell. Therefore, we performed BCECF fluorescence imaging experiments in combination with the whole-cell patch clamp technique, to control the membrane potential of the HEK cells and thus the driving force for H + into and out of the cell.…”

Depletion of plasma membrane–associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg2+, spermine, and pH

“…Such variability could arise from the differences in resting membrane potentials among HEK cells or differences in VSP expression levels. HEK293 cell resting membrane potentials have been estimated to be in the −50 to −30 mV range ( 55 – 58 ). To investigate whether depolarizing shifts in the resting membrane potential will increase VSP activity, transfected HEK cells were cultured in 25.3 m m KCl–containing RPMI medium for 24 h before recordings were made ( Fig.…”

Evidence for functional GABAA but not GABAC receptors in mouse cone photoreceptors

“…GABA receptors could also contribute through their influence on the synaptic cleft pH, as proposed for those of horizontal cells (Liu et al, 2013), through their permeability to HCO3 -. Changes in intracellular Clmay also affect release, either through the modulation of calcium channels (Thoreson et al, 2000;Babai et al, 2010) or through changes in osmotic tension (Chavas et al, 2004). values of the "plateau" current measured at +26 mV (F) or the tail-current measured after a depolarization at +6 mV (G) for rods (grey symbols when spherule is present, n = 13; orange symbols for rods for which no spherule could be seen at the end of the recording but identified as rods through their external segment, n = 11), putative rods (cyan symbols, for cells considered as putative rods due to their soma appearance prior to patching, n = 5), cones (green symbols, presence of a pedicle, n = 11) or putative cones (red symbols, cells for which no pedicle could be seen at the end of the recording, but with a soma appearance suggestive of a cone prior to recording, n = 8).…”