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Published in final edited form as: Curr Opin Biotechnol. 2019 Feb 16;58:100–107. doi: 10.1016/j.copbio.2019.01.004

Ion channel engineering for modulation and de novo generation of electrical excitability

Hung X Nguyen 1, Nenad Bursac 1,*
PMCID: PMC6697248  NIHMSID: NIHMS1519404  PMID: 30776744

Abstract

Ion channels play essential roles in regulating electrical properties of excitable tissues. By leveraging various ion channel gating mechanisms, scientists have developed a versatile set of genetically-encoded tools to modulate intrinsic tissue excitability under different experimental settings. In this article, we will review how ion channels activated by voltage, light, small chemicals, stretch, and temperature have been customized to enable control of tissue excitability both in vitro and in vivo. Advantages and limitations of each of these ion channel-engineering platforms will be discussed and notable applications will be highlighted. Furthermore, we will describe recent progress on de novo generation of excitable tissues via expression of appropriate sets of engineered voltage-gated ion channels and discuss potential therapeutic implications.

Graphical abstract

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Introduction

Ion channels are pore-forming transmembrane proteins that enable rapid passage of inorganic ions down their electrochemical gradients into or out of the cell. Since ions are charge carriers, ion channels play pivotal roles in regulating transmembrane potential (Vm) – the voltage difference across cell membrane. At rest, the balanced flow of positive and negative ions through different channels and transporters sets up a stable baseline Vm, called resting membrane potential (RMP). In eukaryotes, RMP varies among different cell types, with more hyperpolarized (negative) values often characterizing electrically excitable cells, including neurons, myocytes, endocrine cells, as well as some plant cells. Upon receiving a stimulus of sufficient strength, the membrane potential of an excitable cell becomes depolarized above a defined threshold to activate voltage-gated ion channels, resulting in fast rise of Vm (depolarization), followed by a slower recovery (repolarization) back to resting state. This consistent, short-lasting fluctuation in membrane voltage (called “action potential”, AP) is the basis of membrane excitability [1] that underlies various cellular processes including neuronal signal propagation, muscle contraction, neurotransmitter release, and hormone secretion.

The opening and closing of ion channels are regulated (or gated) by various types of stimuli, including transmembrane voltage [2], binding of extracellular of intracellular molecules [3,4], light [5,6], stretch [7], and temperature [8]. In recent years, ion channel researchers have adapted these gating mechanisms to develop and optimize powerful methods for controlling cellular excitability in vitro and in vivo. Such endeavors have been further guided via valuable insights from comprehensive electrophysiological characterizations, newly published high-resolution channel structures, and molecular dynamics simulations. In this review, we will discuss recent advances in the engineering and optimization of a diverse set of ion channel families for modulation and de novo generation of electrical excitability. Not only have these sophisticated tools been utilized for mechanistic studies of excitable cell biology, but recently, they have also opened doors to potential therapies for excitable tissue disorders.

Engineering light-gated ion channels for spatiotemporal control of excitability

Since the first proof-of-concept study demonstrating light-based activation of neurons via heterologous expression of Chanelrhodopsin-2 (ChR2) [9] (Figure 1A), an expanding list of optogenetic actuators have been developed [10], aided by the solved 2.3 Å crystal structure of the chimeric ChR1/ChR2 channel [11] and the discovery of new microbial opsins with diverse kinetics, ion selectivity, and spectral properties [12–14]. Specifically, the E123T/A mutation in the “ChETA” variants was shown to destabilize the protonated state of the retinal Schiff base (RSB) and speed up channel deactivation after light-off, enabling high-frequency stimulation up to 200 Hz [15]. Other fast-deactivating variants have been discovered via de novo sequencing of algal transcriptomes [12], including “CheRiff” [16] and “Chronos” – the fastest ChR homolog reported to date [12]. Conversely, for applications that require subthreshold membrane depolarization over prolonged periods of time, it is more desirable to extend channel opening duration without continuous light exposure. This has been achieved via the engineering of step-function opsin (SFO) variants [17,18], by single or double mutations of the C128/D156 residue pair. Once illuminated with brief excitation pulse, these variants remain in the conducting state for minutes or upon termination by red-shifted light.

Figure 1.

Figure 1.

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Ion channel engineering for modulation of electrical excitability. (A) Light-gated ion channel ChR2 for activation of excitability with high spatiotemporal resolution. (B) Magnetothermal stimulation of heat-activated channel TRPV1. (C) Ultrasonic stimulation of mechanosensitive channel MscL. Only Na+ and Ca2+ ions are shown here for simplicity. (D) Inhibitory chimeric ligand-gated channel PSAM-GlyR, with ligand-binding domain (brown) engineered from nAChR and pore domain (green) from chloride-conducting channel GlyR.

Traditionally, to inhibit cell excitability by light-driven membrane hyperpolarization, researchers have employed Halorhodopsin (NpHR) [19] or Archaerhodopsin (Arch) [20]. However, since these chloride and proton pumps rely on active transport of one ion per absorbed photon, they exhibit low light sensitivity and require high-intensity illumination. This has been addressed by converting cation-conducting ChR variants into chloride selective channels via several rounds of mutagenesis that involved remodeling of the electronegative into electropositive ChR pore [21,22] and complete elimination of the proton conductance pathway. The resulting highly anion-conducting ChR (ACR) variants (iC++ [23] and iChloC [24]) have large photocurrent, high light sensitivity, and reversal potential similar to calculated Nernst potential for chloride. Furthermore, introducing the SFO mutation C128A into iC++ results in bistable inhibitory channel (SwiChR++), enabling prolonged neuronal silencing [23]. The inhibitory optogenetics toolbox has been further expanded with the recent discovery of naturally occurring anion-conductive ChR homologs from different cryptophyte species [13,14], with the notable variant “ZipACR” capable of inhibiting individual spikes during neuronal firing at 50 Hz. Furthermore, high-resolution crystal structures of both designed (iC++ [25]) and naturally-occurring (GtACR1 [26]) ACRs have recently been reported, revealing various insights into mechanisms involved in channel gating, anion-conducting pathway, pH red-shifting of ACR (iC++ G220S/S295A) and engineering of FLASH, a fast ACR with fast-off kinetics and high anion selectivity [25].

Beside controlling the on and off kinetics of light-gated channels, developing red-shifted ChR variants has been of particular interest as they would enable all-optical interrogation of neuronal circuits when used in combination with genetically encoded calcium and voltage indicators (GECI and GEVI) [27]. Specifically, the ChR variants that activate at spectral peaks above 600 nm would facilitate in vivo deep tissue stimulation due to significantly reduced light absorption and scattering at these wavelengths [28]. Since the first red-shifted ChR homolog (VChR1) was reported in Volvox carteri [29], several new variants have been discovered and developed with improved spectral and photocurrent properties. Of note are “Chrimson” [12] – most red-shifted ChR known to date with spectral peak at 590 nm and “bReaChES” [30] – chimeric ChR channel bearing ChETA mutation with strong photocurrent and high spiking fidelity.

In addition to playing essential roles in the interrogation of neural circuits, engineered microbial opsins can be employed for in situ and ex vivo assessment of functional integration between stem cell-derived neuronal grafts and host nervous system for potential cell therapy applications [31]. Moreover, these light-gated channels have been finding use in the emerging field of cardiac optogenetics [32], where they are utilized for high-throughput in vitro cardiac electrophysiology assays, mechanistic examination of arrhythmia dynamics, and proof-of-concept studies demonstrating their translational potential for cardiac rhythm management.

Engineering ion channels for deep tissue control of excitability

Despite the progress in design of red-shifted ChR variants, their action spectra still fall short of the 650–1350 nm (near-infrared, NIR) window for maximal tissue penetration. To circumvent this limitation and enable minimally invasive control of deep tissue excitability in cell-specific fashion, researchers have been developing additional genetically-encoded tools that permit optical, magnetic, and ultrasonic stimulation [33]. Specifically, lanthanide-doped upconverting nanoparticles (UCNPs) were produced to convert low-energy NIR light into high-energy visible emission light for activation of microbial opsins expressed in deep tissue [34]. The emission spectra of UCNPs can be tuned to match the action spectra of available opsin variants. In particular, UCNPs with blue emission can be combined with ChR2 for neuronal excitation, while those with green emission are compatible with NpHR and Arch for neuronal silencing [34]. Alternatively, heat-inducing properties of infrared (IR) light can be used to activate the thermosensitive channels from the transient receptor potential (TRP) family, such as the snake TRPA1 channel [35] and the vampire bat TRPV1-L channel [36], to achieve neuronal stimulation with single-cell resolution.

Deep tissue activation can be also achieved using static or alternating magnetic fields applied to other thermosensitive channels from the TRP family. Specifically, low-radiofrequency (0.1–1MHz) alternating magnetic fields in combination with magnetic nanoparticles (MNPs) have been employed to generate thermal energy and activate TRPV1 channels, triggering membrane depolarization [37] (Figure 1B). In freely moving animals, MNP injection can be obviated by utilizing a single-component magnetogenetic system in which TRPV4 channel is fused with the paramagnetic protein ferritin [38]. Furthermore, a single mutation (I679K) in the S6 region of TRPV1, converts the channel from cation-conducting to chloride-selective thus permitting magnetic inhibition of excitability [39]. Alternatively, magnetic fields can be used to activate the two-pore mechanosensitive potassium channel TREK-1 in which high-affinity binding to magnetic particles coated with anti-His antibodies has been enabled by inserting a 6-histidine repeat into the S1–S2 extracellular loop of the channel [40].

Another modality for activating mechanosensitive channels is via low-pressure ultrasound, which also provides minimal invasiveness and deep tissue penetration. For example, neuron-specific expression of the mechanosensitive channel TRP-4 has allowed ultrasonic activation of neurons in the presence of gas-filled microbubbles to modulate behavioral output of C. elegans [41]. The need for microbubbles can be obviated using the engineered version of bacterial large-conductance mechanosensitive channel MscL [42,43] (Figure 1C) in which channel expression and membrane trafficking are optimized and channel mechanosensitivity is amplified via the I92L or G22S mutations. This has enabled ultrasound-triggering of spike trains in cultured neurons with millisecond precision and spiking fidelity up to 5 Hz [42]. As the biophysical properties and molecular structure of MscL are well-characterized, further channel modifications could be made to fine-tune gating kinetics, ionic conductance, and mechanosensitivity. The more recent discovery of the mammalian mechanosensitive Piezo channel family [44] could also expand the described sonogenetics toolbox. Calcium selectivity of Piezo1, in particular, can be employed for ultrasonic activation of the downstream gene transcription via control of calcium influx [45]. Recently reported high-resolution molecular structure of Piezo1 [46,47] is expected to facilitate the future engineering of these channels for ultrasonic modulation of excitability.

Engineering ligand-gated ion channels for chronic modulation of excitability

While light-based systems (optogenetics) provide unmatched spatiotemporal resolution in controlling tissue excitability, the use of ligand-activated systems (chemogenetics) is more suitable for applications that require non-invasive manipulation of excitability over sustained periods of time (up to hours or days) [48]. Recent advances in this area have led to development of versatile chemogenetic tools for in vivo control of excitability including engineered G-protein coupled receptors (GPCRs), most notably the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) systems and engineered ligand-gated ion channels (LGICs). Since GPCR-based tools rely on indirect manipulation of cell excitability via endogenous signaling pathways, they will not be the discussed here (for a recent comprehensive review of DREADDs, refer to [49]). In contrast, engineered LGICs allow powerful activation or inhibition of excitability via direct control of ion channel current [50].

One LGIC-based approach relies on the activation of Cys-loop ion channels upon binding to the natural antiparasitic drug ivermectin (IVM). In a heteromeric glutamate-gated chloride channel from Caenorhabditis elegans (GluCl αβ), multiple mutagenesis rounds were performed to eliminate its native glutamate sensitivity, increase IVM sensitivity, enhance channel expression level, and improve its trafficking to the plasma membrane [51]. Such engineered channels (e.g. GluCl v2.0) have enabled IVM-induced silencing of stem cell-derived sensory neurons in vitro and modulation of neuropathic pain thresholds in vivo [52]. More potent effects on membrane potential can be attained by engineering a human glycine receptor (α1 GlyR) to take advantage of its large unitary channel conductance and homomeric channel structure. Specifically, introduction of the F207A mutation at the glycine-binding site effectively eliminates native glycine sensitivity while single mutation at the pore domain (A288G) renders the channel highly sensitive to IVM [53]. Furthermore, introducing three additional mutations in transmembrane domain TM2 can convert engineered GlyR from anion-permeable to cation-permeable channels, opening doors for the development of IVM-sensitive excitatory tools [54].

Additional approaches to LGIC design focus on constructing chimeric channels made from the engineered ligand binding domain (LBD) from the α7 nicotinic acetylcholine receptor (α7 nAChR) and the ion pore domain (IPD) from other Cys-loop receptors [55] (Figure 1D). Mutagenesis at the ligand binding pocket of the LBD and screening against a focused chemical library has led to identification of three LBD mutants (W77F, Q79G, and L141F) that display selectivity for additional, structurally distinct ligands. Other mutations (Y115F, Q139G, and L141S) can be used to diminish channel’s sensitivity for the endogenous ligand acetylcholine. Combining these two groups of mutations has led to construction of Pharmacologically Selective Actuator Modules (PSAMs) that recognize their cognate Pharmacologically Selective Effector Module (PSEM) agonists but are not responsive to physiological levels of acetylcholine. Fusion of PSAMs to the IPD of cation-conducting 5HT3 channel generates excitatory chimeric channels that depolarize membrane in the presence of cognate PSEMs. Conversely, combining PSAM with IPDs of chloride-selective GlyR and GABA C channels yields inhibitory channels for PSEM-induced sustained silencing of excitability. The ability of the PSAM/PSEM system to modulate excitability in opposite directions has been utilized for in vivo bidirectional control of pain responses [56], long-term memory consolidation [57], and blood pressure [58]. Further advances in chemogenetic technologies are expected to spawn novel and improve existing therapies for neurological disorders.

Engineering voltage-gated ion channels for de novo generation of excitable tissues with customized properties

In addition to direct manipulation of excitable cells, exogenous expression of appropriate ion channel combinations could confer excitability in naturally unexcitable cells [59]. Indeed, we have previously demonstrated the generation of electrically excitable and actively conducting human embryonic kidney cells (HEK-293s) via stable co-expression of three genes encoding for a voltage-gated sodium channel (Nav1.5), an inward-rectifier potassium channel (Kir2.1), and a gap junction channel (Cx43) [60]. These cells, named “Ex-293”, have since been utilized for in vitro examination of basic electrophysiology, pharmacology, and channelopathies [61,62]. Similar approach has been applied by others to convert HEK-293 cells into spontaneously spiking cells for high-throughput screening of genetically-encoded voltage indicators [63] and blue light-activated excitable cells for all-optical pharmacological screening of ion channel modulators [64] (Figure 2A).

Figure 2.

Figure 2.

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Combining engineered ion channels for de novo generation of electrical excitability. (A) Generation of excitable cells for all-optical pharmacological screening of ion channel inhibitors [64]. Activation of CheRiff channel with blue light depolarizes membrane potential to the threshold required for Nav1.7 opening, which then triggers action potential (AP) firing. Change in membrane potential is reflected as a change in fluorescence signal of QuasAr2. Excitability is reduced or abolished in the presence of Nav1.7 inhibitors. (B) Generation of electrically excitable and actively conducting tissues [66]. Following electrical stimulation (pulse sign), cell on the left fires AP due to activation of BacNav channel. AP then propagates to the cell on the right due to the presence of Cx43 gap junction channel formed between the two cells. In both examples, excitable cells maintain a hyperpolarized membrane potential at rest via Kir2.1 expression.

De novo generation of excitable primary cells is a more technically challenging feat as genes encoding for mammalian Nav channels are too large (>6kb) to be packaged into lentivirus or adeno-associated virus for efficient and stable transduction. This limitation has been solved by use of bacterial Nav channels (BacNav) [65] as their significantly smaller gene size (<1kb) allows co-packaging with other ion channel genes in the same viral vector, paving the way for engineering of more complex electrophysiological phenotypes. Use of lentiviruses expressing BacNav, Kir2.1, and Cx43, has enabled conversion of various primary human cell types into excitable cells with robust electrical phenotype [66] (Figure 2B). Furthermore, in an in vitro model of interstitial fibrosis, engineered excitable fibroblasts rescued impaired cardiac conduction to healthy levels, suggesting the potential for use in cell-based cardiac therapies [66].

Since the discovery of the first BacNav [65], significant progress has been made in understanding their structure [67,68], gating [69], and pharmacology [70]. Moreover, novel BacNav orthologs have been discovered and functionally characterized, revealing diverse voltage dependencies and gating kinetics [71]. Such expanding knowledge of the BacNav toolbox could be utilized, in combination with computational modeling and high-throughput screening methods, to facilitate optimization and customization of electrical properties in engineered cells. Mutating the D60 residue of NavSheP channel, for instance, can yield variants with diverse biophysical properties and allow identification of mutants that yield improved conduction velocity in engineered cells [66]. With further advances, direct expression of engineered BacNav in a tissue- and environment-specific fashion may allow development of targeted gene therapies to address loss of excitable cells due to degenerative disease or injury.

Conclusions

The powerful and fast-expanding field of ion channel engineering has adapted various principles of channel gating to enable bidirectional control of excitability for probing tissue function, dissecting biological mechanisms, and potential therapeutic applications. Different flavors of the technology have been developed to target specific questions and experimental needs. In particular, light-gated ion channels with fast deactivation kinetics are most beneficial for applications that require high spatiotemporal resolution and specific firing patterns. For deep tissue applications, ultrasonic, magnetic, or infrared stimulation of mechanosensitive or heat-activated channels can improve access to cells and avoid the need for invasive use of visible light guides. On the other hand, engineered ligand-gated ion channels are best suited for chronic noninvasive modulation of excitability in spatially extended cell populations. In addition to controlling intrinsic excitability, exogenous expression of engineered ion channels can be utilized for potential cell or gene therapies to generate de novo excitable tissues. Through progress in the field, several recurring themes have emerged, including the adaptation of ion channels from microorganisms, the transferability of functionally similar domains between channels from the same family, and the conversion between excitatory and inhibitory channel actuation via mutations along ion conducting pathway. We anticipate that these ion channel engineering principles, together with recent advances in structural biology and the expansion of protein sequence databases, will fuel future development of ion channel-based tools for basic electrophysiology studies and potential clinical applications.

Highlights.

  • Engineering microbial opsins for control of excitability with high spatiotemporal resolution.

  • Deep tissue control of excitability using infrared, ultrasonic, and magnetic stimulation.

  • Engineering ligand-gated ion channels from Cys-loop receptor family for chronic modulation of excitability.

  • Engineered prokaryotic sodium channels for the generation of primary excitable tissues as potential cell therapies.

Acknowledgement

This work was supported by the National Institutes of Health [grant numbers HL095069, HL104326, HL132389, HL126524, and HL126193].

Footnotes

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