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Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels.

ABSTRACT: Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca2+/CaM-regulation of CaV1 and NaV1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into CaV1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca2+/CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca2+/CaM signaling to individual targets.

SUBMITTER: Niu J

PROVIDER: S-EPMC6156082 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels.

Niu Jacqueline J Dick Ivy E IE Yang Wanjun W Bamgboye Moradeke A MA Yue David T DT Tomaselli Gordon G Inoue Takanari T Ben-Johny Manu M

eLife 20180910

Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (sta ...[more]

PMID: 30198845

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Project description:Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.

Project description:Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.

Project description:The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 microM) to high (1,024 microM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0. 5 11.2 +/- 0.3 microM at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of approximately 0.97 for Ca2+i approximately 100 microM. Increasing Ca2+i further to 1,024 microM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 microM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.

Dataset Information

Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels.

Publications

Allosteric regulators selectively prevent Ca<sup>2+</sup>-feedback of Ca<sub>V</sub> and Na<sub>V</sub> channels.

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