Ca ²⁺ channel nanodomains boost local Ca ²⁺ amplitude

Abstract

Local Ca 2+ signals through voltage-gated Ca 2+ channels (Ca V s) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single Ca V channel and the Ca 2+ signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca 2+ measurements. Here, we exploited Ca 2+ -dependent inactivation of Ca V channels as a nanometer-range Ca 2+ indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca 2+ amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of Ca V nanodomains, as diffusion-restricted environments that amplify small Ca 2+ fluxes into enormous local Ca 2+ concentrations. This Ca 2+ tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca 2+ load.L ocal Ca 2+ signals increase the information capacity of signaling within a cell, by enabling short-range signals to operate independently of Ca 2+ events elsewhere throughout the cell (1, 2). These local Ca 2+ signals are the conduit that drives diverse activity-dependent events, such as synaptic transmission (3), neural plasticity (4-6), and cardiac excitation-contraction coupling (7). However, with regard to the Ca 2+ amplitude within nanometers of individual Ca 2+ channels, our knowledge is based predominantly on diffusion theory, which predicts ∼100 μM Ca 2+ signals per picoampere flux at a distance of ∼10 nm from the pore (8-11). Although often quoted, this theory has been difficult to verify experimentally because diffusible Ca 2+ indicators report space-averaged [Ca 2+ ], and thus lack the spatial resolution needed for selective nanodomain reporting. Two recent efforts to overcome this limitation used voltage-gated Ca 2+ channel (Ca V )-tethered fluorescent Ca 2+ indicators to isolate nanodomain signals (12, 13), but were met with unexpected difficulties: Ca V -tethered indicators were unresponsive in the presence of high concentrations of intracellular 1,2-bis(oaminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca 2+ buffer that preserves Ca 2+ elevations within tens of nanometers of active channels but eliminates it elsewhere (8-10). A lack of response under these conditions would arise if the majority of tethered channels were silent, raising the concern that fluorescence changes observed under milder Ca 2+ buffering (12, 13) may represent the response of indicators tethered to silent channels, which nonetheless eavesdrop on Ca 2+ from active channels nearby. To overcome this fundamental limitation, we used a reverse strategy, whereby Ca 2+ /calmodulin-dependent inactivation (CDI) of channels themselves serves as a nanometer-range indicator of [Ca 2+ ]; a sensitive ionic readout that is unaffected by the pres...

A Human Ventricular Myocyte Model with a Refined Representation of Excitation-Contraction Coupling

“…However, most models of Ca 2þ -dependent inactivation (CDI) are not necessarily based on the cascade of molecular events of the CDI of LCCs (24)(25)(26)(27). On the other hand, the Hinch model gives a Ca 2þ concentration at the Ca 2þ -binding site for CDI, which is consistent with the widely accepted theoretical estimation of [Ca 2þ ] near the ion channel exit (25,26,(28)(29)(30). If the estimation of [Ca 2þ ] at the ion channel exit is realistic, the shortening of AP evoked by increasing the [Ca 2þ ] o (31,32) might be reproduced in a mathematical model through the CDI mechanism.…”

Surface dynamics of voltage-gated ion channels

“…128 Therefore, VGCC activation-driven fluctuations in intracellular calcium concentrations have been the subject of intense investigations. 30,104,128,129 VGCCs are composed of a pore-forming a1 subunit, an intracellular b subunit and extracellular a2d subunits. 43 While the role of g subunits in a functional VGCC is not entirely clear, it is thought that they act mainly as auxiliary subunits of AMPA receptors.…”

“…137 Given these important functions of VGCC in intracellular and intercellular signaling, the positioning of VGCC in respect to calcium-sensitive intracellular molecules is of high relevance for the signaling capacity of calcium fluxes. In most models, the positioning of calcium channels has been assumed to be fixed in place to maximize the signaling capacity of local intracellular calcium domains of opened channels without crosstalk to neighboring entry sites, 101,129 but there is an accumulating body of evidence to suggest otherwise. 41,123 In addition, the fast action of intracellular calcium buffers and calcium-binding proteins support this picture.…”

“…41,123 In addition, the fast action of intracellular calcium buffers and calcium-binding proteins support this picture. 49,129 Changes in the conductivity, steady-state inactivation by the interaction with SNARE proteins such as syntaxin and SNAP 25, 14,15 as well as calmodulin mediated calcium-dependent inactivation/facilitation 30,99 are considered as the self-possessed properties of the channel that determine its signaling capacity. As proposed by Tadross et al, 129 the induced calcium domain of one VGCC (Ca V 1.3) seems to be restricted to a few nanometres -but with a much higher amplitude than assumed before.…”

“…49,129 Changes in the conductivity, steady-state inactivation by the interaction with SNARE proteins such as syntaxin and SNAP 25, 14,15 as well as calmodulin mediated calcium-dependent inactivation/facilitation 30,99 are considered as the self-possessed properties of the channel that determine its signaling capacity. As proposed by Tadross et al, 129 the induced calcium domain of one VGCC (Ca V 1.3) seems to be restricted to a few nanometres -but with a much higher amplitude than assumed before. 74,104 Hence, the signaling capacity of VGCCs seems to be maximised by their local action and implies a signaling precision within a membrane domain of < 100nm.…”