Mechanisms Underlying Influence of Bioelectricity in Development
There is a large body of evidence that indicates that potassium also plays an important role in cell-cycle regulation (Urrego et al., 2014). Treating cells with potassium channel blockers can block proliferation (DeCoursey et al., 1984; Amigorena et al., 1990; Lee et al., 1993), and this is partially due to potassium’s role in calcium movement. The potassium gradient hyperpolarizes the cell membrane, driving calcium entry into the cell and thus potassium can regulate the cell cycle via the calcium mediated pathways described above (Urrego et al., 2014). However, the role of potassium channels in hyperpolarizing the cell membrane is not the only mechanism by which they influence the cell cycle. Multiple potassium channels including KV1.3, KV3.1, and KV10.1 impact the cell cycle even when they are modified to prevent ion permeation, suggesting that they may interact with cell signaling via a mechanism that is independent of potassium conduction (Downie et al., 2008; Millership et al., 2011; Cidad et al., 2012; Urrego et al., 2014).
While chloride appears to play a less important role in the cell cycle than calcium and potassium, the chloride channel ClC3 plays a role in cell cycle regulation in nasopharyngeal carcinoma cells and in glial cells, with disruption of CIC3 inhibiting cell proliferation (Habela et al., 2008; Xu et al., 2010). ClC3 regulates cell volume, and chloride efflux is necessary to cause the reduction in volume seen in mitotic cells (Habela et al., 2009). A reduction in cell volume via efflux of salt and water is important for mitosis, and cells that are forced to maintain a larger volume take longer to divide (Habela and Sontheimer, 2007). When chloride flux is blocked, cells cannot reduce their volume prior to mitosis, and this leads to a delay in cell division. Intracellular chloride levels have also been shown to directly regulate the cell cycle by regulating the expression level of p21. Loss of chloride leads to an upregulation of p21 which in turn leads to a downregulation of CDK2 and cell cycle arrest at the G1/S cell cycle checkpoint (Miyazaki et al., 2008; Shiozaki et al., 2011). Changes in proliferation because of disruption of ion channel function in individual cells would impact the size of a whole tissue.
The role of ion channels in proliferation as well as cell death pathways is one of the primary reasons that ion channel mutations are common in cancer cells. The role of ion channels in cancer has been reviewed extensively (Lang and Stournaras, 2014; Prevarskaya et al., 2018). The variety of ion channels linked to cancer suggest that ion channels play an important role in regulating the cell cycle.
Ions are important for both the establishment of cell polarity and the progression of cell migration (Campetelli et al., 2012). In human bone osteosarcoma U2OS cells, it has been observed that many calcium channels, including those that regulate the ER stores of calcium, tend to concentrate at the rear end of polarized cells (Huang et al., 2015). Disruption of a variety of calcium channels using drugs that targeted transient receptor potential channels (TRPC), calcium release activated channels (CRAC), or store-operated calcium entry (SOCE) channels, all led to a decrease in cell polarization (Huang et al., 2015). Disruption of STIM via knockdown or expression of a dominant-negative form of STIM, similarly reduced cell polarization (Huang et al., 2015). While the mechanism by which calcium channels regulate cell polarity is unclear, at the immune synapse calcium organizes actin filament formation (Hartzell et al., 2016). Actin plays an essential role in planar cell polarity, and it is possible that calcium impacts cell polarity by regulating actin dynamics.
The establishment of cell polarity is essential for the migration of cells. Calcium plays roles in cell migration in vivo as well as in cell culture. For example, blocking spontaneous calcium waves in the developing chick feather bud disrupts normal cell migration leading to malformed feather buds (Li et al., 2018). Calcium is similarly important for the migration of zebrafish primordial germ cells (PGCs) (Blaser et al., 2006). In these PGCs it was found that calcium levels increased at the front of migrating cells and this increase was necessary for proper migration (Blaser et al., 2006). Blaser et al. hypothesize that this increase in calcium may activate acto-myosin contraction, directing cell migration (Blaser et al., 2006). Cell migration is important for morphogenesis of several structures. If individual cells cannot migrate properly due to inhibition or loss of ion channel function, cells will not be in the right place at the right time to send or receive developmental signals and the tissue would not develop normally. In addition, lack of effective migration could prevent the correct number of cells from reaching their proper location in a tissue. Therefore, disruption of ion channels could impact the development of a structure by hindering cellular migration.
Other ions contribute to establishment of cell polarity. Inhibition of Na,K-ATPase or treatment with a sodium ionophore in epithelial cells leads to a loss of cell polarity, suggesting that regulation of sodium is important for establishment of cell polarity (Rajasekaran et al., 2001). Overexpression of RhoA GTPase rescues this loss of cell polarity (Rajasekaran et al., 2001). Rho organizes actin and tight junctions in polarized epithelia (Nusrat et al., 1995), so this suggests that sodium is important for this Rho-dependent cell polarization pathway.
While ion channels and ions regulate many essential cellular processes long known to be important for development, there is a more recent hypothesis that ion channels may directly regulate the morphogen signaling pathways to coordinate development. Within multiple organisms, loss of ion channel function is associated with disruptions in the BMP signaling pathway, the Notch signaling pathway, the Wnt signaling pathway, and the Hedgehog signaling pathway.
BMPs are signaling proteins that are essential for the development of organs and tissues, regulating proliferation, apoptosis, and differentiation. Disruption of various ion channels leads to defects in BMP signaling, suggesting that bioelectrical signaling may help regulate this pathway. In mouse bone marrow mesenchymal stem cells (BMSCs), a disruption of BMP signaling and differentiation was found upon knockout of the calcium channel Orai1 (Lee et al., 2016). This disruption of BMP signaling could be rescued by expression of a constitutively active BMP receptor (Lee et al., 2016). Orai1 is a CRAC that helps regulate ER calcium, so loss of BMP signaling upon Orai1 knockout suggests that ER calcium may help regulate BMP signaling in mouse BMSCs. Another channel involved in ER calcium regulation, sarcoendoplasmic reticulum calcium transport ATPase (SERCA) plays a role in the regulation of BMP signaling. In the D. melanogaster air sac primordium (ASP), downregulation of SERCA leads to a decrease in BMP/Dpp signal transduction (Huang et al., 2019). Similar impacts on BMP/Dpp signal transduction were found upon knockdown of the voltage-gated calcium channel genes straightjacket (stj) and cacophony (cac) (Huang et al., 2019). Interestingly this disruption of BMP/Dpp signal transduction in the ASP was also found upon knockdown of the calcium binding proteins Syt4 or synaptobrevin (Syb) (Huang et al., 2019). Syt4 and Syb are both involved in vesicle trafficking, suggesting that proper BMP/Dpp signaling in this system may require vesicle trafficking mediated by calcium (Huang et al., 2019).
Potassium channels also play a role in BMP/Dpp signaling. Kir2.1 is an inwardly rectifying potassium channel that when disrupted in humans is associated with morphological differences as part of Andersen-Tawil Syndrome. Kir2.1 function is associated with proper BMP signaling in multiple organisms. In mice, Kir2.1 knockout leads to abnormal limb development, craniofacial defects, and a significant reduction in Smad 1/5/8 phosphorylation indicating that Kir2.1 is required for BMP pathway functioning in mammals (Belus et al., 2018). Similar craniofacial defects occur in developing frogs upon loss of Kir2.1 function (Adams et al., 2016). In the Drosophila wing disc, loss of function of Irk2, the Drosophila ortholog of Kir2.1, reduces downstream phosphorylation of Mad and BMP/Dpp target gene expression (Dahal et al., 2012). The similar developmental disruptions that occur in flies, frogs, and mice upon Kir2.1/Irk2 disruption, suggest that this potassium channel plays a conserved role in development.
In Drosophila loss of Irk2 function disrupts BMP/Dpp secretion dynamics, likely leading to the disruption of BMP/Dpp signaling and defects in wing morphogenesis (Dahal et al., 2012; Dahal et al., 2017). Irk2 disruption also abolishes spontaneous calcium oscillations in the wing, so this impact of Irk2 on BMP/Dpp secretion may be mediated through its impact on calcium (Dahal et al., 2012; Dahal et al., 2017). One potential hypothesis is that Irk2 along with calcium channels or other ion channels regulate depolarization events, which in turn regulate the fusion of BMP/Dpp containing vesicles to the cell membrane (Figure 4). This would explain a potential mechanism by which BMP/Dpp secretion could be regulated, impacting propagation of the downstream BMP signaling pathway. Depolarization of the developing Drosophila wing evokes BMP/Dpp release, supporting this model (Dahal et al., 2017).
Notch signaling is another canonical developmental signaling pathway that is impacted by the disruption of ion channels. Notch signaling is a conserved signaling pathway required for the development of many tissues and organs (Kopan and Ilagan, 2009). The ligands in the Notch pathway are transmembrane proteins rather than secreted ones and thus Notch signaling acts as a short range signal (Kopan and Ilagan, 2009). Notch signaling regulates cell division, cell death, and cell differentiation (Kopan and Ilagan, 2009).
Regulation of ER stores of calcium are important for proper functioning of the Notch signaling pathway (Figure 5). In Drosophila, SERCA, a channel that pumps calcium into the ER, is particularly important for Notch signaling. Disrupting SERCA function in Drosophila S2 cells, in the Drosophila eye, or in the Drosophila larval wing disc, leads to developmental defects consistent with a loss of Notch signaling as well as an accumulation of notch and delta receptors in intracellular vesicular structures away from the cell surface (Periz and Fortini, 1999; Suisse and Treisman, 2019). This accumulation of notch away from the cell surface is also seen in the Drosophila wing disc when Orai is knocked down (Suisse and Treisman, 2019). Orai and SERCA both act to regulate ER calcium levels, suggesting that ER calcium is important for the trafficking of notch or delta (Figure 5). Loss of SERCA functioning in human leukemia cells, also leads to intracellular accumulation of Notch with the Notch1 receptor failing to fully mature, suggesting that this role of ER calcium in Notch signaling may be conserved (Roti et al., 2013). The Notch receptor contains calcium binding EGF-like repeats, and it is possible that when calcium levels in the ER drop, the notch receptor is no longer able to fold correctly leading to its accumulation within the ER or a failure to traffic correctly to the cell surface (Rand et al., 1997).
Notch signaling also regulates bioelectricity (Figure 5). In human embryonic kidney 293 (HEK293) cells and in myocytes upregulation of Notch signaling has been associated with an increase in cytosolic calcium and decreases in potassium flux (Khandekar et al., 2016; Song et al., 2020). In the HEK293 cells Notch signaling attenuates the activity of voltage-gated potassium channels while also inducing clustering of Stim channels, leading to an influx of calcium into the cytoplasm from the ER (Song et al., 2020). These results suggest that Notch signaling may both regulate and be regulated by ion channel function.
Wnt Signaling, another important developmental signaling pathway is also regulated by ion channel function. In the Drosophila wing disc disruption of SERCA, an ER calcium channel, causes E-Cadherin to be retained in the ER (Suisse and Treisman, 2019). This causes β-catenin/Arm, which binds to E-Cadherin, to be sequestered in the ER and unable to participate in signaling, leading to downregulation of Wnt signaling (Suisse and Treisman, 2019) (Figure 6). This downregulation of Wnt signaling was also found upon disruption of the ER calcium regulating channel Orai, suggesting that ER calcium plays an important role in Wnt signaling (Suisse and Treisman, 2019) (Figure 6).
While there is evidence that calcium is important for Wnt signaling propagation, potassium and chloride both appear to play even more important roles in this pathway (Rapetti-Mauss et al., 2020) (Figure 6). Potassium regulates the localization of β-catenin impacting Wnt signaling. Inhibition of the potassium channel KCNQ1 downregulates Wnt/β-catenin signaling. This is due to the role of KCNQ1 in regulating the membrane potential. Overactivation of KCNQ1 hyperpolarizes cell membrane while inhibition of KCNQ1 depolarizes the cell membrane (Rapetti-Mauss et al., 2017; Rapetti-Mauss et al., 2020). Inhibition of KCNQ1 and the subsequent depolarization of the membrane inhibits β-catenin from localizing to the cell membrane attenuating Wnt/β-catenin signaling (Rapetti-Mauss et al., 2017; Rapetti-Mauss et al., 2020).
Chloride signaling, too, has been associated with regulation of Wnt/β-catenin signaling. Disruption of CTFR, the chloride channel associated with cystic fibrosis, leads to an increase in intracellular pH (Strubberg et al., 2018). This change in pH enhances the interaction between the Wnt signaling receptors Disheveled and Frizzled leading to an increase in Wnt signaling (Strubberg et al., 2018; Rapetti-Mauss et al., 2020). This increase in Wnt signaling upon CTFR disruption may be one of reasons why cystic fibrosis is associated with abnormal lung development and increased risk of gastrointestinal cancer (Neglia et al., 1995; Larson and Cohen, 2005).
The hedgehog signaling pathway family members, including sonic hedgehog (Shh), desert hedgehog (Dhh), and Indian hedgehog (Ihh) all play an essential role in embryonic patterning and development (Choy and Cheng, 2012). In the hedgehog pathway, the ligands act as secreted morphogens facilitating longer range signaling (Choy and Cheng, 2012). While evidence suggests that calcium can regulate BMP and Notch signaling, in contrast hedgehog signaling appears to primarily act upstream of calcium, regulating calcium oscillations. Recent studies suggest that calcium may play an important role in the execution of the hedgehog signaling pathway. In zebrafish, disruption of intracellular calcium release from the ER via RyRs resulted in abnormal neural tube patterning which was attributed to a loss of Shh-dependent gene expression (Klatt Shaw et al., 2018). RyR function was found to be specifically important for the Shh ligand receiving cells, indicating a role for calcium in Shh signal transduction (Klatt Shaw et al., 2018). Multiple other studies have also implicated spikes of calcium in the execution of Shh induced signaling in Xenopus, mouse, and rat embryos and cell lines (Osawa et al., 2006; Heo et al., 2007; Belgacem and Borodinsky, 2011). While the exact mechanism by which Shh induced calcium oscillations modulate gene expression is unclear, it has been suggested that Shh-mediated induction of calcium activates ERK signaling which in turn changes gene expression (Osawa et al., 2006). Shh was also found to mediate calcium oscillations in chick feather buds (Li et al., 2018). In the chick feather bud it was found that Shh could induce expression of the calcium channels Connexin-43 and Stim1 to induce calcium oscillations which were important for the migration of the cells in the bud (Li et al., 2018).
In the developing Drosophila wing (wing disc), hedgehog signaling and ion channel control of Vmem mutually reinforce each other (Emmons-Bell and Hariharan, 2021) (Figure 7). A Vmem reporting dye shows a stripe of depolarized cells can be found near the anterior/posterior (A/P) boundary, with the depolarization becoming more restricted to the anterior side of the boundary over time (Emmons-Bell and Hariharan, 2021). Disrupting degenerin epithelial Na+ channels (DEG/ENaC) prevents depolarization of that stripe of cells (Emmons-Bell and Hariharan, 2021). Upon both an increase or decrease in hedgehog signaling via activation of a temperature sensitive hedgehog allele or a constitutively active Cubitus interruptus (Ci) allele, the expression levels of the DEG/ENaC channel Rpk and the Na+/K+ ATPase subunit ATPα were found to change (Emmons-Bell and Hariharan, 2021). These expression levels of Rpk and ATPα correspond with a change in Vmem, suggesting that hedgehog signaling regulates the Vmem pattern of cells within a tissue via regulation of ion channel expression (Emmons-Bell and Hariharan, 2021). Conversely, reducing expression of Rpk or ATPα using wing-specific RNAi reduces Hh signaling (Emmons-Bell and Hariharan, 2021). Direct modulation of Vmem via optogenetics regulates smoothened membrane localization, suggesting that bioelectricity regulates Hh signaling while also being regulated by it (Emmons-Bell and Hariharan, 2021). This suggests that Hh signaling and Vmem mutually reinforce each other (Figure 7).
Taken together, these studies suggest that all of the major canonical signaling pathways can be regulated by bioelectricity. Notch and Hh signaling regulate ion channel signaling while also being regulated by changes in bioelectricity. BMP and Wnt signaling both lie downstream of ion channel function and the activity of these pathways can be modulated by changes in bioelectricity.
Salvador Mafe, Michael Levin, and Javier Cervera have proposed that membrane potential could regulate transcription directly (Cervera et al., 2020; Levin, 2021). Thus, bioelectric signals could coordinate cellular outcomes between several cells within a developing tissue. In this model, bioelectric fields would control development on a tissue wide scale.
It is becoming increasingly evident that ion channel function and the Vmem pattern in tissues is essential for development. Because ion channels play roles in nearly every essential developmental process including cell death, proliferation, cell polarity, migration, and regulation of the canonical developmental signaling pathways, this raises the exciting possibility that cells within tissues may use bioelectrical signaling as a high-level mechanism to coordinate the complex process of development of a tissue. While we described mechanisms on an individual cell level, in multicellular organisms, cells are working within a tissue. Thus, the contribution of ion channels to proliferation, apoptosis, cell migration, and signaling would impact the morphogenesis of a whole tissue or structure. For example, if the ion channels that impact proliferation and migration are inhibited during palatogenesis, the correct number of cells would not migrate to the palate shelves and would not proliferate adequately for palate shelves to reach one another and fuse at the midline, which would result in a cleft palate.
Calcium appears to be the major player in bioelectrical signaling. In each of the cellular processes which ion channels help regulate, calcium channels play the largest role of all of the ion channel types: calcium is the primary ion that acts in ion channel mediated regulation of cell death, working in both the ER related and mitochondria related cell death pathways, calcium acts at nearly every stage in the cell cycle to regulate proliferation, calcium channels are required for the establishment of cell polarity and for cell migration, and calcium plays a role in BMP, Notch, Wnt, and Hh signaling. ER regulating calcium channels are specifically required for many of these processes with SERCA, Stim, or Orai having been identified as necessary for nearly all of these cellular processes (Figure 2–Figure 6). Remarkably, these ER-calcium regulating channels are also necessary for the propagation of the calcium oscillations that occur spontaneously in developing tissues (Narciso et al., 2015; Ohno and Otaki, 2015; Restrepo and Basler, 2016; Balaji et al., 2017; Li et al., 2018), providing a potential link between calcium oscillations and these calcium-regulated cellular processes. The near universal existence of calcium oscillations in developing tissues paired with the known roles of calcium in multiple cellular processes and pathways raises intriguing possibilities for the role of calcium in guiding development. Calcium oscillations provide a mechanism by which cells could communicate in detailed ways. Oscillations contain many encoded variables—such as frequency, amplitude, and rate of change—that could each potentially carry information, allowing cells to fine-tune communication through subtle changes in oscillatory properties. Could it be that a variety of ion channels contribute to development by converging on regulation of intracellular calcium?
Cellular concentrations of one ion can influence concentrations of other ions. If the activity of one type of ion channel is impaired, concentrations the ion it conducts are altered, but concentrations of other ions can be changed as well. For example, membrane potential impacts cytoplasmic calcium levels. This is due to the high number of voltage-gated calcium channels that are able to respond to changes in Vmem by opening or closing and allowing or stopping calcium flux (Catterall, 2011). Many other channel types—including potassium, sodium, and chloride channels—are calcium sensitive and open upon calcium binding and lead to changes in Vmem. Calcium-activated potassium channels in particular have been associated with calcium induced changes in Vmem (Lazzari-Dean et al., 2019). This feedback between calcium levels and Vmem allows both properties to mutually regulate each other.
The known roles of calcium oscillations and Vmem in development suggest a model in which these factors can be used for communication. A potential model can be imagined in which cells within growing tissues have varying Vmem values depending on their location within the tissue and the propagation of calcium oscillations. Because mechanical forces can induce changes in Vmem and calcium oscillations it is possible that the individual forces on each cell—which depend on the cell’s placement within a tissue—may help regulate this bioelectrical signaling. In turn, these bioelectrical signals could regulate the proliferation, death, cell polarity, and migration of each cell while also regulating the canonical developmental signaling pathways, ultimately guiding each cell to differentiate at the proper time and place to form the adult organism. Because bioelectrical signals such as calcium oscillations can encode multiple different variables, information could be fine-tuned to each cell. Whether a cell decides to divide, proliferate, die, or differentiate could depend not only on an overall level of a particular ion but on the combination of Vmem and calcium oscillation frequency or amplitude. Cells within tissues are interconnected via a vast network of gap junctions, so the bioelectrical state of each cell could in turn regulate the bioelectrical states of cells nearby. This model would suggest a mechanism by which cells are able to communicate rapidly and dynamically in response to perturbations such as damage during development.
Ion channels have emerged as important regulators of cell death, cell cycle regulation, cell polarity, migration, and canonical developmental signaling pathways. While the field of developmental bioelectricity is rapidly growing, there are still many questions to be answered.
It has only recently been discovered that ion channels play a role in well-established developmental signaling pathways such as BMP, Wnt, and Notch. How do the important molecular signaling pathways and ion channel signaling intersect? Ion channels clearly impinge upon BMP, hedgehog, notch, and wnt signaling pathways, but it is not well understood whether these effects are due to direct regulation of these pathways or due to downstream effects on cell cycle, cell death, or cell migration. While some research suggests that the role of ion channels in signaling is via a direct mechanism such as regulation of the secretion or trafficking of components of the BMP and notch signaling pathways (Dahal et al., 2017; Suisse and Treisman, 2019), more studies need to be done specifically investigating these potential mechanisms and whether these roles are conserved in development.
Another question in the field is whether the role of ion channel signaling in development is mediated primarily through impacting the transmembrane potential or through impacts on the spontaneous calcium oscillations that occur in developing organisms. As previously mentioned, both calcium oscillations and transmembrane potential are intrinsically related and influence each other. However, more work needs to be done to understand whether each property influences different aspects of development or whether both properties work within the same pathway. Much of the recent work in the field of developmental bioelectricity has focused on the patterns of Vmem across developing tissues and how these patterns might instruct development. However, while it is clear that transmembrane potential patterns across tissues are an important regulator of multiple cellular processes, there is evidence that the more slight but rapid changes in Vmem that occur via the propagation of calcium oscillations also play an important role in developing tissues. The discovery that many tissues spontaneously propagate calcium transients and waves, raises the possibility that cells could use ionic signaling to communicate across greater distances like neurons. There is evidence that ion channels may impact secretion dynamics of cell signaling pathways and that developing tissues propagate calcium waves. Are these calcium waves regulating morphogen secretion similar to the way action potentials regulate neurotransmitter release in neurons? Recent work in the Drosophila air sac primordium suggests that such a mechanism may be at work in cell types not traditionally thought of as excitable (Huang et al., 2019). In most tissue types, cells are connected via a vast network of gap junctions, enabling such changes in calcium to be used to communicate across tissues.
More work needs to be done to investigate whether similar regulation of morphogens via calcium oscillations occurs in other cell and tissue types. With new tools in optogenetics and calcium sensing now readily available and improving, this research is now possible.
Another unanswered question in the field is how the transmembrane potential and calcium oscillations are regulated and coordinated on a tissue wide scale. If cells are using the transmembrane potential and calcium waves to communicate, then they must still be able to sense their position within a tissue to adopt the correct Vmem. What upstream information allows cells to properly set their transmembrane potential and propagate calcium oscillations? Because many ion channels are sensitive to mechanical stresses, one hypothesis is that mechanical forces between cells within a tissue may guide ionic signaling. Mechanical forces play an important role in development, and mechanosensitive ion channels have been associated with cell processes such as development of cell polarity and migration (Mammoto and Ingber, 2010; Huang et al., 2015; Canales Coutino and Mayor, 2021). In fact, mechanical stressors have been found to induce calcium waves in many cell types and tissues (Godin et al., 2007; Tsutsumi et al., 2009; Narciso et al., 2015; Restrepo and Basler, 2016). Another possibility is that some of the well-established developmental signaling pathways may also shape calcium oscillations. The hedgehog signaling pathway is upstream of calcium waves, suggesting an alternative mechanism by which ion gradients may be regulated (Osawa et al., 2006; Belgacem and Borodinsky, 2011; Klatt Shaw et al., 2018; Li et al., 2018). More work needs to be done, however, to fully understand how calcium oscillations and Vmem patterns are established and regulated as tissues grow.
While there is a rising understanding of the role of ion channel function in each of the key developmental cellular processes of proliferation, cell death, migration, and molecular cell signaling, less is understood about how bioelectrical signaling may regulate these processes all together and how the bioelectrical patterns and each of these processes interconnect across a tissue. For example, oscillations in calcium play a role in progression of the cell cycle, induction of cell death, and activation of cell migration. How does the cell distinguish between these signals? When cytosolic calcium rises how does the cell know whether to divide, die, send a molecular signal, or migrate? One possibility is that the cell responds to narrow ranges of Vmem changes and calcium levels, with information potentially encoded within the frequency or amplitude of calcium oscillations. The resting membrane potential of a given cell in a tissue along with the active genes in each cell type, could dictate how easily calcium influx is induced and to what degree Vmem changes in response, which in turn could regulate whether the cell responds by dividing, dying, migrating, or propagating a developmental signaling pathway. For example, a specific cell that is already in a relatively depolarized region within a tissue would need less calcium influx to reach a specific calcium threshold than a cell in a relatively hyperpolarized region. However, calcium influx into the cell in the hyperpolarized region to reach the same final calcium threshold would result in a final amplitude with larger magnitude.