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Voltage-gated sodium and calcium channels are distinct evolutionarily-related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino-acid sequences within the selectivity filter. By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and a Na+-permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity.
Voltage-gated sodium (NaV) and calcium channels (CaV) are members of an evolutionarily-related ion channel superfamily that function in essential physiological processes. The NaV channels are responsible for initiation and propagation of action potentials (AP) in excitable cells, while CaV channels transduce voltage-changes to the influx of Ca2+ ions, a second messenger process that tunes excitability and supports synaptic neurotransmission, excitation-contraction coupling, and gene regulation (
). In mammals, these channels are multi-subunit complexes encoded by distinct genes. At the molecular level, the pore-forming subunit of the two channel families share a similar pseudo-tetrameric architecture with four homologous domains linked together by intracellular segments. Each domain contains a voltage-sensing domain formed by four transmembrane helices (S1-S4), and two helices (S5-S6) that line the central ion permeation pathway or the channel pore. The pore contains a selectivity filter (SF) that determines ion selectivity. Given this broad similarity, understanding structural and molecular mechanisms that confer ion selectivity is of fundamental biological importance.
From an evolutionary perspective, CaV channels are thought to have emerged and diversified in early unicellular eukaryotes in conjunction with the increased importance of sophisticated Ca2+ signaling mechanisms (
). In a recent issue of the Journal of Biological Chemistry, Guan and colleagues studied ion channels in the heart of the giant pond snail (Lymnaea stagnalis), where APs are driven by both Na+ and Ca2+ currents, yet only a single CaV gene appears to be expressed. They uncovered an intriguing mechanism involving alternative splicing of a CaV3 gene, a homolog of the mammalian low voltage-activated T-type channel, which switches its preference from Ca2+ to Na+ ions. The authors leveraged emerging deep learning-based structure prediction approaches and electrophysiology to identify the molecular basis of this process (
Extensive study over the past fifty years has identified core features responsible for the ion selectivity of voltage-gated ion channels. Classical biophysical studies point to a scheme where the pore domain contains two or more high-affinity binding sites for the specific ion (
). Once bound, coulombic interactions between pairs of ions permit robust flux of the ion despite high-affinity binding. These expectations were largely substantiated by structural studies which revealed the atomic arrangement of the selectivity filter (
). The SF is formed by two pore helices (P1-P2) from each homologous domain. This results in a channel pore with specific dimensions and distinct amino-acid sequences that line the ion permeation pathway. For CaV channels, the signature sequence is a ring of four negatively charged residues, typically glutamates (‘EEEE’), that line the narrow SF (
). For NaV channels, the SF is composed of an aspartate-glutamate-lysine-alanine or ‘DEKA’ sequence. The additional aspartate residue is typically absent. Like other Ca2+ channels, the Lymnaea CaV3 channel contains a ring of negatively charged residues (‘EEDD’) and D[+1] residue (
). In the snail heart, the predominant LCaV3 channel isoform includes exon 12a. Inclusion of this exon increases Na+ permeability. By contrast, the skeletal muscle variant of LCaV3 includes exon 12b, which increases Ca2+ permeability (
). In their recent work, the authors integrated computational structural prediction using AlphaFold with extensive electrophysiological analysis to gain insights on how structural changes outside of the classical selectivity filter can impact ion permeation. In the predicted CaV3 models from L. stagnalis, the exon 12a channel possesses a lysine residue (K) which is hypothesized to form a salt bridge with the D[+1] residue (Fig 1). In channel variants with exon 12b, an alanine (A is present in place of the lysine and is therefore incapable of forming a salt bridge (Fig 1). Guan and colleagues propose that the presence of the lysine residue neutralizes the DII D[+1] residue and change the overall local charge density in the outer pore vestibule. When the D[+1] residue is present, (e.g. with exon 12a), the Ca2+ ion is coordinated by the carboxylate groups of the outer D[+1] and the SF aspartate/glutamates. When a Na+ ion enters the pore, it is repelled by this Ca2+, while incoming Ca2+ ions knock off the chelated Ca2+ ion into the inner pore of the channel. With the D[+1] charge neutralized, channels then allow influx of Na+ ions. Of note, a similar multi-ion permeation mechanism has been proposed for the prokaryotic Bac (bacterial) NaV channels.
To test their hypothesis, Guan and colleagues mutated the key lysine residue in the LCaV3 exon12a variant into alanine and estimated changes in Na+ permeability. Indeed, the peak Na+ current size with the LCaV3-exon12a KA mutant was significantly reduced in comparison to the wild-type LCaV3-exon12a variant, indicating reduced Na+ permeability. Conversely, replacement of the A residue of LCaV3-exon 12b with a lysine led to increased Na+ currents. Analysis of relative permeability ratios also confirmed this trend. To further illustrate this mechanism, the authors introduced charge-neutralizing mutations at the DII D[+1] residue of both LCaV3 and human CaV3.2. In both cases, removal of the charged residue resulted in increased monovalent permeability.
Overall, this study provides new insights into both biophysical and evolutionary mechanisms that determine ion selectivity for voltage-gated channels, a long-standing physiological problem. In mammals, alternative splicing of Ca2+ channels is increasingly recognized as a versatile mechanism for fine tuning various channel properties including subcellular localization and gating. The present study illustrates that splicing can also impact Ca2+ permeability, effectively allowing a Ca2+ channel to become a de facto Na+ channel. What remains unknown, however, is the physiological and evolutionary benefit of this atypical splicing-dependent mechanism, in place of a dedicated NaV gene. It is possible that alternative splicing may be further regulated either developmentally or in response to other stimuli. Another possibility is that the Na+ versus Ca2+ permeability of LCaV3 may have distinct effects on shaping action potentials versus tuning excitation-contraction coupling in the Lymnaea heart. Biophysically, the finding that domains outside of the ion permeation pathway can tune the channel selectivity has important implications. A dizzying array of genetic mutations in nearly all CaV and NaV channels have been linked to a wide range of human diseases; it is possible that some of these mutations may impact channel selectivity, leading to an imbalance in ion homeostasis. Excitingly, this study also highlights the potential utility of deep learning-based computational structure prediction approaches to generate new hypotheses to address long-standing physiological problems (
). Combining in silico predictions with carefully designed electrophysiological studies could substantially aid structure-function analysis of ion channel regulation. Even still, experimental structural determination using, for example, cryo-electron microscopy will be essential to fully substantiate possible structural mechanisms.
Hille, B. (2001) Ion channels of excitable membranes, 3rd ed., Sinauer Associates, Sunderland, MA
Cav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter.