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T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s. [ABSTRACT FROM AUTHOR]

Abstract: The limited choice and poor performance of red-emitting calcium (Ca2+) indicators have hampered microfluorometric measurements of the intracellular free Ca2+ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca2+ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602nm peak excitation/emission) conjugated to BAPTA and having an additional NH2 linker arm. The low-affinity variant (K D,Ca ∼30μM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca2+ transients. While Calcium Ruby is a mitochondrial Ca2+probe, its conjugation, via the NH2 tail, to a 10,000MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca2+ probe with an affinity matched to microdomain Ca2+ signals. As an example, we show depolarization-evoked Ca2+ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca2+ measurements. [Copyright &y& Elsevier]

T-type calcium channels (the CaV3 channel family) are involved in defining the resting membrane potential and in neuronal activities such as oscillations and rebound depolarization. Their physiological roles depend upon the channel activation and inactivation kinetics. A fast inactivation that stops the ionic flux of calcium in tens of milliseconds has already been described in both native and heterologously expressed channels. Here, using HEK 293 cells expressing the rat CaV3.1 channel and whole-cell voltage clamp, we investigate an additional inactivation process, which can be distinguished from the previously described fast inactivation by its slow time course of recovery from inactivation (τ= 1 s) and by its sensitivity to external calcium. Steady-state slow inactivation is voltage dependent around the resting membrane potential (the potential of half-inactivation (V0.5)=−70 mV, slope factor= 7.4 mV) and can reduce the calcium current by up to 50%. Near resting potential, the slow inactivation displays a half-time of induction of tens of seconds. The slow inactivation therefore modulates the availability of T-type calcium channels depending upon recent cell history, providing a mechanism to store information in a time scale of seconds. [ABSTRACT FROM AUTHOR]

Abstract Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (α1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for α1G; 7 ms for α1H; and > 10 ms for α1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the α1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of α1G and α1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of α1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of α1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling. [ABSTRACT FROM AUTHOR]

Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca2+] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.