Voltage-gated Ca2+ (Ca(V)) channels are central to the biology of excitable cells, and therefore regulating their activity has widespread applications. We describe genetically encoded molecules for inducibly inhibiting Ca(V) channels (GEMIICCs). GEMIICCs are derivatives of Rem, a Ras-like GTPase that constitutively inhibits Ca2+ currents (I(Ca)). C terminus-truncated Rem(1-265) lost the ability to inhibit I(Ca) owing to loss of membrane targeting. Fusing the C1 domain of protein kinase Cgamma to yellow fluorescent protein (YFP)-Rem(1-265) generated a molecule that rapidly translocated from cytosol to plasma membrane with phorbol-12,13-dibutyrate in human embryonic kidney cells. Recombinant Ca(V)2.2 and Ca(V)1.2 channels were inhibited concomitantly with C1(PKCgamma)-YFP-Rem(1-265) membrane translocation. The generality of the approach was confirmed by creating a GEMIICC using rapamycin-dependent heterodimerization of YFP-FKBP-Rem(1-265) and a constitutively membrane-targeted rapamycin-binding domain. GEMIICCs reduced I(Ca) without diminishing gating charge, thereby ruling out decreased number of surface channels and voltage-sensor immobilization as mechanisms for inhibition. We introduce small-molecule-regulated GEMIICCs as potent tools for rapidly manipulating Ca2+ signals in excitable cells.

Voltage-dependent calcium-channel β subunits (Ca V β) strongly modulate pore-forming α 1 subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P o ). Despite their central role, it is unclear whether binding of a single Ca V β, or multiple Ca V βs, to an α 1 subunit governs the two distinct functions. Conventional experiments utilizing coexpression of α 1 and Ca V β subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1 : 1 stoichiometry by covalently linking Ca V β 2b to the carboxyl terminus of α 1C (Ca V 1.2), creating α 1C ·β 2b . Recombinant L-type channels reconstituted in HEK 293 cells with α 1C ·β 2b supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed α 1C ·β 2b fully restored channel trafficking to the plasma membrane. Co-transfecting Ca V β 2a with α 1C ·β 2b had little further impact on function. To rule out the possibility that fused Ca V β 2b was interacting in trans with neighbouring α 1 molecules, α 1C ·β 2b was cotransfected with α 1B (Ca V 2.2), and pharmacological block with nimodipine showed an absence of α 1B trafficking. These results establish that association of a single Ca V β with a pore-forming α 1 subunit captures the functional essence of HVA calcium channels, and introduce α 1 -Ca V β fusion proteins as a powerful new tool to probe structure-function mechanisms.

Astrocytes express four types of Cl(-) or anion channels, but Ca(2+)-activated Cl(-) (Cl(Ca)) channels have not been described. We studied Cl(-) channels in a morphologically distinct subpopulation ( approximately 5% of cells) of small (10-12 micro m, 11.8 +/- 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, -57.1 +/- 4.0 mV, polarized to -72.7 +/- 4.5 mV with BAPTA-AM, an intracellular Ca(2+) chelator, and depolarized to -30.7 +/- 6.1 mV with thapsigargin, which mobilizes Ca(2+) from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl(-) reversal potential, which was blocked by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn(2+), by I(-) (10 mM), and by chlorotoxin (EC(50) = 47 nM). With conventional whole-cell clamp, NPPB- and Zn(2+)-sensitive currents became larger with increasing [Ca(2+)](i) (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca(2+) sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive Cl(Ca) channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive Cl(Ca) channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte.