Articles in the popular media often show neurons and muscle cells launching miniature flashes of lightning when they are stimulated. In real cells, these processes are less glossy but still fascinating. Movements, thoughts, feelings, memories, sensations and more are made possible by ions moving in and out of cells through narrow pores in proteins called ion channels, which open and close in response to various stimuli (such as a change in voltage or membrane tension, or a molecule binding to the ion-channel protein). Ion channels are embedded in the cell membrane, and the ionic currents passing through them change the voltage across the membrane, creating an electrical signal that can be propagated to the interior of the cell or to other cells (Hille, 2001).
Like all other proteins, ion channels are the product of natural selection (Anderson and Greenberg, 2001), and we can learn about their evolution by comparing the genes that code for similar ion channels in different species (Moran and Zakon, 2014). An important property of an ion channel is its selectivity, which determines the types of ions that can pass through it. Sodium ion channels have been identified in bacteria and other prokaryotes, and some of these channels can be mutated to gain selectivity for calcium, but until recently no native calcium ion channels had been observed in prokaryotes.
Now, in eLife, Katsumasa Irie of Nagoya University and colleagues – including Takushi Shimomura as first author – report the first identification of a native prokaryotic calcium ion channel in Meiothermus ruber, a species of bacteria that lives in hot springs (Shimomura et al., 2020). They also propose a selectivity mechanism that is different from that found in calcium ion channels in eukaryotes.
By aligning multiple DNA sequences of sodium and calcium channels from prokaryotes, Shimomura et al. also reconstructed a phylogenetic tree to show how sodium and calcium channels in different species evolved from a common ancestor (Figure 1). This revealed a new branch of the phylogenetic tree containing what they have called ancestor-like sodium channels (which are homologous to contemporary sodium channels). The selectivity filters of the newly identified calcium channel and the ancestor-like sodium channels have sequences similar to the one predicted for an ancestor of a sodium channel found in bacteria (Liebeskind et al., 2013) and, remarkably, to the sequences found in a number of calcium channels from mammals.
The sequences for two homologues of the prokaryotic sodium channels were then used to synthesize the corresponding channels in mammalian and insect cells, so that their electrical properties could be measured. The channel based on M. ruber showed a high selectivity for ions with a charge of 2+: indeed, its selectivity for Ca2+ was ~200 times greater than its selectivity for Na+. It is generally thought that the selectivity of calcium channels is due to the presence of aspartates, which are negatively charged, in the selectivity filter (Catterall and Zheng, 2015). However, when the M. ruber calcium channel was mutated to eliminate an aspartate in this region, the channel retained most of its selectivity for Ca2+ ions.
On the other hand, the channel from Plesiocystis pacifica, a bacterial species that lives in soil, was three times more selective for Na+ ions than it was for Ca2+ ions. Moreover, the flow of Na+ ions through this channel could be blocked by high concentrations of extracellular Ca2+ ions. Furthermore, the blocking effect of Ca2+ could be enhanced by adding an alanine residue, which has no charge, to the selectivity filter, despite the presence of three negatively charged amino acids in this region.
These findings suggest that the selectivity of these two ion channels may not solely depend on the presence of negative charges in the filter.
The results from M. ruber and P. pacifica prompted Shimomura et al. to investigate the molecular basis of Ca2+ selectivity in these channels. They found that if the selectivity filter of the M. ruber calcium channel was mutated to be the same as the P. pacifica sodium channel, this new channel lost its Ca2+ selectivity and behaved like a non-selective channel. However, when the P. pacifica sodium channel was mutated to be the same as the M. ruber calcium channel, it demonstrated levels of calcium selectivity similar to those observed in M. ruber.
M. ruber and P. pacifica have different amino acids at positions 4 and 6 in their selectivity filters. Shimomura et al. found that changing the glycine at position 4 in M. ruber to a serine or an aspartate reduced the Ca2+ selectivity by a factor of almost 25, and wiped out any selectivity for ions with a single positive charge (such as Na+). The small and flexible glycine residue in position 4 would make the pore wider and facilitate the entry of ions with a charge of 2+, such as Ca2+. The conservation of this glycine residue in some eukaryotic calcium channels suggests that it might be part of a more general calcium-selectivity mechanism. Structural analysis of the new channels will shed light on this possibility.
Finally, the latest work indicates that voltage-dependent Ca2+ signaling is more ancient than previously thought, and the presence of calcium channels in prokaryotes suggests the possibility that Ca2+-selective channels in eukaryotes are not derived from sodium channels, as previously postulated. The new family of channels described by Shimomura et al. opens a tantalizing window into a vast evolutionary landscape that we are just beginning to grasp.
Phylogeny of ion channels: clues to structure and functionComparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 129:17–28.https://doi.org/10.1016/S1096-4959(01)00376-1
Deciphering voltage-gated Na+ and Ca2+ channels by studying prokaryotic ancestorsTrends in Biochemical Sciences 40:526–534.https://doi.org/10.1016/j.tibs.2015.07.002
BookIon Channels of Excitable MembranesSunderland, MA: Sinauer Associates.
- Version of Record published: February 25, 2020 (version 1)
© 2020, Suárez-Delgado and Islas
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The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.
Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.