CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation.
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ABSTRACT:
SUBMITTER: Hudmon A
PROVIDER: S-EPMC1343528 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand-like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.
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Project description:Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.
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Project description:Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent "junctional" sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca(2+) signaling complexes in many cell types. We examined the possibility that some Ca(2+) signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca(2+) sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the alpha1 or alpha2 isoform of the Na(+) pump catalytic (alpha) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that alpha2(f)GCaMP2, like native Na(+) pumps with alpha2-subunits, sorted to PM domains that colocalized with subjacent S/ER; alpha1(f)GCaMP2, like Na(+) pumps with alpha1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca(2+) signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca(2+) channel-mediated Ca(2+) entry after S/ER unloading with cyclopiazonic acid (in Ca(2+)-free medium). When cytosolic Ca(2+) diffusion was markedly restricted with EGTA, however, only alpha2(f)GCaMP2 detected the local, store-operated Ca(2+) channel-mediated Ca(2+) entry signal. Thus, alpha1 Na(+) pumps are apparently excluded from the PM microdomains occupied by alpha2 Na(2+) pumps. The jS/ER and adjacent PM may communicate by Ca(2+) signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca(2+) signals in other subPM microdomains and signals associated with other organelles.
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Project description:CaMKII plays a critical role in decoding calcium (Ca2+) signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ signals in spines remain elusive. Here, we measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each pulse during repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. Thr286 phosphorylation slows the decay of CaMKII and thus lowers the frequency required to induce spine plasticity by several fold. In the absence of Thr286 phosphorylation, increasing the stimulation frequency results in high peak mutant CaMKIIT286A activity that is sufficient for inducing plasticity. Our findings demonstrate that Thr286 phosphorylation plays an important role in induction of LTP by integrating Ca2+ signals, and it greatly promotes, but is dispensable for, the activation of CaMKII and LTP.
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