IDRs in Cross-Membrane Transport: Regulation of Ion Channels and Transporters, Mechanistic Studies Made Possible by NMR and Computational Methods

Introduction

The classical “one structure–one function” paradigm, experimentally confirmed by Christian B. Anfinsen and earning him the Nobel Prize in 1972, does not adequately explain the structure-activity relationships of many proteins. As this limitation became clear, the need for a new concept emerged. By the late 20th century, intrinsically disordered proteins (IDPs) were introduced (Wright and Dyson, 1999). Together with proteins containing intrinsically disordered regions (IDRs), they now appear to constitute a substantial portion of eukaryotic genomes. Estimates indicate (Tomp and a, 2002) that (i) 30–40% of residues in the eukaryotic proteome are predicted to be in disordered regions, (ii) approximately 70% of eukaryotic proteins contain at least one disordered segment, often longer than 30–50 amino acids, and (iii) about 10% of proteins are fully disordered, while hybrid proteins combining structured domains and IDRs represent the majority. Structurally, IDPs and disordered regions of hybrid proteins do not adopt a single stable fold. Instead, they are best described through energy landscapes characterized by broad energy wells with numerous local minima separated by low energy barriers (Dyson and Wright, 2005).

Biochemical, structural, and dynamic studies of intrinsically disordered proteins (IDPs) initially concentrated on soluble proteins, as these systems are technically more tractable than their membrane counterparts. This emphasis reflects the well-established influence of water’s polarity on the amino acid composition and behavior of soluble IDPs. By contrast, the transmembrane (TM) segments of membrane proteins reside within the hydrophobic core of the lipid bilayer, formed by nonpolar aliphatic chains. To avoid energetically unfavorable exposure of backbone polar groups, amide protons and carbonyl oxygens must satisfy intramolecular hydrogen bonding requirements, thereby enforcing ordered secondary structure within membrane-spanning regions of integral membrane proteins (IMPs). Converging evidence now indicates that the membrane-water interface is a critical locus for intrinsic disorder and disorder–order transitions with significant physiological consequences. Furthermore, disordered cytoplasmic or extracellular domains, as well as flexible loops, frequently connect TM helices or β-strands, positioning IMPs as major contributors to the class of hybrid proteins that combine folded domains and intrinsically disordered regions (IDRs). Despite their prevalence, structure-activity relationships in hybrid integral membrane and membrane-associated proteins remain comparatively underexplored. For example, a query of the DisProt database (https://disprot.org/, accessed 1 October 2025) shows that only ∼320 of ∼3,200 entries include the keyword “membrane,” accounting for approximately 10% of the collection.

To help address this gap and stimulate broader interest in this protein class, this manuscript examines how IDRs regulate cross-membrane transport, with primary attention to ion channels and transporters. To start with, I briefly review established functions of IDRs in soluble, membrane-associated proteins, noting that comprehensive coverage of their roles in vesicular and non-vesicular transport is available elsewhere (Sigrist and Haucke, 2023). This short discussion is included to expose a more extensive experimental database present for and our better understanding of IDRs within soluble membrane-associated proteins in comparison to integral membrane counterparts. I follow up and spend most of the time on the primary objective of this review - I highlight the importance and, by showing what has been done, try to stimulate general interest in defining the roles of IDRs in ion channels and transporters. At the end, I discuss how nuclear magnetic resonance (NMR) methods, often integrated with computational approaches, enable atomic-level characterization of structure and dynamics in flexible regions of large, membrane-spanning complexes.

IDR roles in membrane transport

Hybrid membrane and membrane-associated proteins often contain long stretches of disordered polypeptide chain, frequently exceeding 30 residues, which confer unique properties enabling spatiotemporal control and tight regulation of membrane transport. These hybrid proteins exploit the complementary advantages of folded domains, such as high-affinity and selective binding, alongside the distinct attributes of intrinsically disordered regions. IDRs are generally characterized by binding promiscuity, a feature that can serve multiple functions and helps optimize the limited intracellular space. They are also highly susceptible to post-translational modifications, making them particularly well-suited for roles in signal transduction across membranes.

Beyond signaling, IDRs influence membrane protein dynamics in diverse ways. Some modulate rates of internalization and recycling within cellular organelles, while others drive the formation of dynamic condensates through liquid-liquid phase separation (LLPS), potentially clustering associated binding partners. At the membrane-water interface, IDRs provide flexible scaffolds that enable bending, tethering, and remodeling of membrane surfaces through interactions with other biomolecules (Box 1).

Before addressing the roles of IDRs in ion channels and transporters, I first highlight recent mechanistic studies illustrating IDR functions in soluble, membrane-associated proteins. These studies demonstrate how IDRs in coat proteins, adaptors, and tethering factors act as flexible scaffolds to (i) sense membrane curvature and (ii) reshape membrane architecture and content. This short overview underscores that even among soluble membrane-associated proteins, far more amenable to structural and mechanistic analysis than integral membrane proteins, only a fraction has been experimentally characterized to define IDR structure-activity relationships (as summarized in Table 1 with reported entries in DisProt database, which are only listed when the experimental data provided goes beyond “missing electron density in X-ray structures” statement). A comprehensive review of the roles of disordered regions and proteins in orchestrating vesicular and non-vesicular membrane dynamics is provided by Sigrist and Haucke (2023).

TABLE 1. Several examples of key trafficking proteins containing IDRs (with DisProt references whenever available).

IDRs as membrane curvature sensors, modulators and organelle-specific targeting agents

Zeno et al. (2018) demonstrated using a tethered vesicle assay that the IDR domains of AP180, Neurofilament-M, Epsin1, and Amphiphysin1 sense positive membrane curvature at levels comparable to structured curvature-sensing domains and can synergistically amplify their sensitivity. Importantly, they identified a novel curvature-sensing mechanism unique to disordered proteins, driven by the entropy of the amino acid chain rather than molecular crowding at membrane surfaces (Stachowiak et al., 2012). In a subsequent study, the same group showed that Amphiphysin1 integrates its structured, crescent-shaped BAR domain with a disordered C-terminal region to cooperatively sense and promote membrane curvature extension (Zeno et al., 2019).

Yuan et al. (2023) explored how attractive and repulsive interactions within IDRs influence membrane bending direction. As predicted by modeling, repulsive interactions among disordered regions drive convex (positive) curvature, whereas attractive interactions promote concave (negative) curvature, forming membrane-bound, liquid-like condensates (Figure 1). Interestingly, in chimeric constructs containing both interaction types, curvature depended on the region’s proximity to the membrane: when the attractive region was closer, condensation amplified steric pressure among repulsive regions, favoring convex curvature; conversely, when the repulsive region was closer, attractive interactions dominated, resulting in concave curvature.

Poveda-Cuevas et al. (2024) employed advanced computational modeling and extensive molecular dynamics (MD) simulations to show that the IDR of the ER-phagy receptor FAM134B enhances membrane remodeling. While IDRs alone can sense curvature, their combination with structured reticulon homology domains (RHDs) intensified remodeling and promoted efficient protein clustering, accelerating budding. These findings were validated experimentally by assessing ER fragmentation in U2OS cells via confocal immunofluorescence microscopy. In a related study, Yorimitsu and Sato (2025) used fluorescence microscopy to demonstrate that IDRs direct protein localization within the ER. For example, Sed4 interacts with Sec16 at ER-exit sites (ERES) to facilitate ER export; loss of this interaction causes Sed4 to redistribute to ER tubules, likely mediated by its luminal IDR.

Utility of IDRs in soluble membrane associated proteins for membrane dynamics and transport.

• Provide flexible scaffolds that enable membrane bending, tethering, and remodeling.
• Exhibit conformational plasticity, allowing them to bridge variable distances and adapt to changing membrane curvature.
• Offer multivalency to cluster cargo - often selected via short linear motifs (SLiMs) - and recruit machinery for budding, fission, and fusion.
• Mediate tethering between organelles at ER-mitochondria and ER-plasma membrane contact sites.
• Organize lipid transfer proteins and ion channels at membrane junctions.
• Act as tunable molecular switches through post-translational modifications (PTMs), making them ideal for processes requiring regulated secretion (e.g., neurotransmitters, hormones).
• Drive liquid-liquid phase separation (LLPS) at membranes, creating nanodomains that concentrate transport machinery; the reversible nature of these condensates enables spatiotemporal control of transport.

Synapsin provides a well-characterized example of a soluble, membrane-associated IDP capable of initiating liquid–liquid phase separation (LLPS). Milovanovic et al. (2018) first showed that synapsin forms liquid biomolecular condensates, either alone or with scaffolding proteins, that trap lipid vesicles. Remarkably, these condensates rapidly disassemble upon phosphorylation by Ca²⁺/calmodulin-dependent protein kinase II, mimicking presynaptic dispersion upon stimulation. Later, Pechstein et al. (2020) confirmed that synapsin-driven phase separation underlies synaptic vesicle clustering in vivo.

An innovative biophysical study of clathrin-mediated endocytosis (CME) revealed that liquid-like assembly of CME initiator proteins Eps15 and Fcho1/2 plays a critical role in vesicle formation. This LLPS-based mechanism ensures efficient vesicle assembly while maintaining effective vesicle release (Day et al., 2021). The dynamic nature of Eps15/Fcho1/2 condensates enables the formation of a highly interconnected interaction network that can undergo extensive remodeling as the endocytic structure matures. Supporting this concept, Kozak and Kaksonen (2022) demonstrated that the yeast homolog of Eps15, Ede1, also forms phase-separated condensates, reinforcing the role of LLPS in CME initiation.

IDRs in ion channels and transporters

This section focuses on how intrinsically disordered regions in integral membrane proteins can facilitate or inhibit transmembrane transport (Box 2).

Gating and permeation control

Maintaining cellular homeostasis requires tight regulation of ion channel and transporter permeability. Several mechanisms achieve this control. In the context of structural disorder, early studies proposed that short terminal IDRs can bind to the channel cavity and block ion flow via a “ball-and-chain” mechanism. This concept was first introduced for the Shaker potassium channel (Hoshi et al., 1990) in the early 1990s. Recent cryo-EM advances have revealed molecular details of this mechanism in the prokaryotic MthK calcium-activated potassium channel. Fan et al. demonstrated that a 17-residue N-terminal region, previously unresolved in X-ray structures, mediates this inactivation. The N-terminal motif of a single subunit appears as an elongated electron density inserted into the open pore of the channel tetramer. This density can be modeled as a transient helix connected to the TM1 helix in high-tilt and medium-tilt conformers of the open-inactivated state (Fan et al., 2020).

Another study shows that conformational changes in disordered regions can modulate ion transport by altering the geometry and dynamics of the selectivity filter or other gates. For example, the intrinsically disordered N-terminal ectodomain (Nterm) of the copper transporter Ctr1 was shown, through molecular dynamics simulations and circular dichroism (CD) experiments, to toggle between distinct conformational states (Figure 2) and regulate Cu(I) permeation (Aupic et al., 2022). In the apo state, the Nterm is unfolded, but Cu(I) binding induces β-sheet formation. Lipid-protein interactions further influence this equilibrium: in the alpha state, stabilized by lipid binding, Met1 from one Nterm monomer interacts with the top Met triad of the selectivity filter. This interaction triggers a transition of one TM2 helix from bent to linear, opening the selectivity filter at both ends of Met triad. These findings suggest that Met1 is critical for Cu delivery from the Nterm to the selectivity filter. Overall, this supports the concept that IDRs can function as dynamic “gates,” not merely passive plugs, for ion permeation.

Recent structural studies have characterized the highly flexible intracellular domain (ICD) of the human α7 nicotinic acetylcholine receptor, a member of the pentameric ligand-gated ion channel (pLGIC) superfamily, also known as Cys-loop receptors. Bondarenko et al. (2022) combined NMR and electron spin resonance (ESR) with Rosetta modeling to determine full-length ICD structures in the resting state (Figures 3a–c). The higher relative intensity of the peaks in the spectrum shown (Figure 3d), the more flexible backbones of the corresponding residues are. The regions with the highest intensities demonstrate the most pronounced local motion, therefore reflecting general IDR dynamics. Approximately 57% of ICD residues reside in highly flexible regions, primarily within a large loop connecting TM helices 3 and 4 whose most mobile segment spans ∼50 Å from the channel axis. Motifs involved in cytoplasmic binding, regulation, and signaling were identified in both helical and disordered regions, underscoring the essential role of ICD conformational plasticity in diverse biological processes. Given the high homology and sequence identity among Cys-loop receptors and the generally dynamic nature of the loop connecting TM3 and TM4 (Langlhofer and Villmann, 2016), this approach offers a powerful strategy for resolving IDR structures in this receptor family - structures that remain inaccessible to X-ray crystallography or cryo-EM.

Dynamic regulatory hubs

The structure-activity relationship of another representative of the pLGIC superfamily, the GABA_A receptor (GABA_AR), was thoroughly investigated by Ye et al. (2021). Using isothermal titration calorimetry (ITC) mapping, they identified an 18-amino-acid motif within the flexible loop connecting TM3 and TM4 of the γ2 subunit’s intracellular domain (ICD) that binds GABARAP proteins, promoting GABA_AR surface stabilization. Clathrin adaptor protein AP2 competes for this binding site, and post-translational modifications (PTMs) within the motif differentially modulate receptor interactions. Specifically, phosphorylation of γ2-GABA_AR Y403 increases affinity for GABARAP while reducing affinity for AP2, shifting the equilibrium from endocytosis toward retention.

Utility of IDRs in ion chanels and transporters.

• Gating and permeation control: IDRs can function as dynamic gates, not merely passive plugs, influencing channel opening and ion flow.
• Regulatory hubs: IDRs frequently harbor post-translational modification sites, adaptor-binding motifs, and lipid interaction regions, enabling channels and transporters to integrate multiple signaling pathways.
• Trafficking and localization: Disordered tails often serve as sorting signals, determining whether channels are retained in the ER, delivered to the plasma membrane, or clustered at synapses.
• Clustering: IDRs promote multivalent interactions and phase separation, facilitating the formation of functional channel nanodomains.
• Gating and permeation control: IDR can act as dynamic gate, besides being just a simple plug.
• Regulatory hubs: IDRs often contain post-translational modification sites, motifs for adaptor binding, and lipid interaction sites that allow channels/transporters to integrate multiple signals.
• Trafficking and localization: disordered tails can act as sorting signals, determining whether channels are retained in the ER, sent to the plasma membrane, or clustered at synapses.
• Clustering: IDRs drive multivalent interactions and phase separation, creating functional channel nanodomains.

Interestingly, protein kinase A (PKA)-dependent phosphorylation within the ICD large loop connecting TM3 and TM4 (specifically at S346) of another superfamily member, the glycine receptor (GlyR) α3 subunit, reduces channel conductance (Moraga-Cid et al., 2020), as does the charge-mimicking mutation S346E. This finding directly links PTMs in IDRs to altered permeation properties of pLGICs.

McKinnon et al. examined the impact of altering the length and composition of the large TM3-TM4 loop in the 5-HT_3A serotonin receptor, another superfamily representative (McKinnon et al., 2012). Voltage-clamp and patch-clamp recordings revealed that although the long loop (>75 amino acids) typical of eukaryotic Cys-loop channels is not essential for receptor assembly or function, its length and composition influence channel expression and desensitization. Cryo-EM structures of 5-HT_3A in saposin discs (mimicking lipid bilayers), including symmetric and asymmetric apo states and an asymmetric agonist-bound state, were determined and validated by MD simulations (Zhan et al., 2021). As expected, ICDs in these models remain disordered and partially unresolved, while unbiased MD simulations support their role in long-range allosteric coupling to the pore.

Transient receptor potential (TRP) channels form a large and functionally diverse superfamily of integral membrane proteins, acting as sensory transducers and polymodal signal integrators. Structural studies of TRPs have advanced significantly, with the landmark cryo-EM structure of TRPV1 in 2013 marking the onset of the “resolution revolution” (Liao et al., 2013). However, highly dynamic regions, such as flexible N- and C-termini, remain absent from available X-ray and cryo-EM structures, limiting detailed molecular understanding of TRP channel dynamics and their integration into cellular signaling networks. To address this gap, complementary techniques such as NMR, which excels at probing dynamics and conformational ensembles, are increasingly employed.

Following this approach, Goretzki et al. determined NMR backbone assignments for the highly flexible N-terminal region of TRPV4 (Goretzki et al., 2022). Human TRPV4, a ubiquitously expressed homo-tetrameric cation channel involved in temperature, osmotic, and mechanical sensing, begins with an intrinsically disordered N-terminus of ∼150 amino acids. Deletion of this IDR significantly reduces channel excitability to agonists, underscoring its critical role in TRPV4 activation and regulation. Building on these assignments, Goretzki et al. characterized the structural ensemble of the TRPV4 IDR and its network of antagonistic regulatory elements using NMR, SAXS (small angle X-ray scattering), HDX (hydrogen-deuterium exchange), and MD simulations (Goretzki et al., 2023). They identified a highly conserved autoinhibitory patch that competes with PIP₂ binding to attenuate channel activity (Figure 4). Further, the same group (Goretzki et al., 2018) demonstrated that the proline-rich region (PRR) at the C-terminus of the IDR interacts with the Syndapin3/PACSIN3 SH3 domain. NMR analysis revealed that the bound PRR adopts a class I polyproline II (PPII) helix which is disrupted by a conserved proline (P128) in cis conformation. SH3 binding rigidifies both the PRR and the adjacent PIP₂-binding site.

Similarly, TRPV1, whose first high-resolution cryo-EM structure lacked disordered regions, was studied by NMR and MD simulations. Its C-terminal domain was characterized as intrinsically unstructured at room temperature, both in the presence and absence of lipids. Elevated temperature and PIP₂ binding induced substantial conformational changes and secondary structure formation, potentially transmitted to the TM domain to sensitize the channel (Raymond et al., 2014). Collectively, these studies demonstrate that IDR structural dynamics are tightly coupled to TRP channel activity, providing critical insights into ion channel regulation.

Voltage-gated inwardly rectifying potassium channel KCNH2 (hERG) provides another example of a flexible linker acting as a regulatory switch. Kang and colleagues characterized the N-terminal cytoplasmic domain (NTD) using solution NMR and demonstrated that residues within its folded PAS domain interact with a peptide derived from the S4-S5 linker, a region critical for channel gating (Li et al., 2010). Subsequent studies revealed that this peptide is intrinsically disordered in aqueous solution, confirming the flexible nature of the linker. Interestingly, in detergent micelles, the peptide adopts a 3 (10)-amphipathic helix, suggesting a potential membrane-interaction patch (Gayen et al., 2012). Cryo-EM structure of a truncated hERG channel lacking the NTD flexible linker shows the voltage sensors (S1-S4) in a depolarized conformation with the channel pore open (Wang and MacKinnon, 2017). Voltage sensors movement is proposed to be transmitted, at least in part, through displacement of S5 helix mediated by the S4-S5 linker, which in this case adopts a rigid, non-helical conformation. An inward and centric displacement of S4 closes the S6 helical gate by compressing the S5 helices. Potentially inhibitory interactions of the linker with N-terminal domain from former studies, however, would be missed in this model due to limitations of the truncated construct.

Na⁺-Ca²⁺ exchangers (NCX): architectural features relevant to intrinsically disordered regions

The studies discussed above are all focused on ion channels. This raises an important question: do analogous examples of IDRs or disorder-order transitions exist in membrane transporters? Compelling evidence in support of this notion emerges from studies of Na⁺/Ca²⁺ exchangers (NCX).

NCX constitute a large and evolutionarily conserved family of plasma membrane transporters that mediate Ca²⁺/Na⁺ exchange across biological membranes and play a central role in Ca²⁺ signaling and homeostasis. Members of this family are found across a broad spectrum of organisms, ranging from bacteria to mammals. Despite sharing a common overall topology and many conserved structural elements, NCX orthologs, isoforms, and splice variants display pronounced differences in ion transport kinetics, regulatory mechanisms, and responsiveness to cellular cues.

The molecular basis for this functional diversity remains incompletely understood. Notably, extensive variation is observed in cytoplasmic regions that are poorly resolved structurally and are predicted to exhibit intrinsic disorder. These regions are thought to participate in regulatory interactions and conformational switching, suggesting that disorder–order transitions may play a key role in shaping NCX function. As reviewed comprehensively by Khananshvili (2022), such dynamic structural features offer a plausible, yet largely unexplored, explanation for the remarkable diversity in NCX regulatory behavior.

Eukaryotic and bacterial NCX share a conserved transmembrane (TM) topology composed of 10 helices connected by a large intracellular loop, which separates the TM domain into two homologous halves (TMs 1–5 and TMs 6–10) at the level of the primary sequence. A key difference between these two groups is that eukaryotic NCX proteins contain an extensive regulatory domain embedded within this intracellular loop (Figure 5), whereas their prokaryotic counterparts lack this feature. The TM domain is responsible for ion exchange and has been visualized through crystal structures of the archaeabacterial exchanger NCX_Mj (Liao et al., 2016; Liao et al., 2012). In contrast, the intracellular domain mediates allosteric regulation of exchanger activity by cytosolic Ca²⁺ and Na⁺, a mechanism thought to underlie the pronounced differences in ion transport rates among NCX isoforms and splice variants. Cryo-EM structures of human cardiac NCX1 (Xue et al., 2023) confirmed that cytosolic Ca²⁺ binds to two calcium-binding domains (CBD1 and CBD2) within the intracellular loop, thereby enhancing transport activity. Conversely, cytosolic Na⁺ inhibits exchanger function by promoting a Na⁺-dependent inactivated state (Figure 5a). Notably, several highly mobile residues (341–365) within the linker connecting the β-hub to CBD1 were omitted from the construct used for cryo-EM analysis. This truncation was implemented to minimize cytoplasmic domain heterogeneity and improve structural resolution. Together with stabilization by a Fab fragment, this approach enabled high-resolution (3.1 Å) determination of the Na⁺-dependent inactivated state in the absence of Ca²⁺. In the presence of Ca²⁺, however, NCX1 remained highly dynamic, necessitating particle classification into at least two distinct populations for lower-resolution models building. These were interpreted as an apo-like state (group I), resembling the Ca²⁺-free structure, and a Ca²⁺-bound active state (group II).

Despite all the advances, the regulatory contributions of the truncated and intrinsically flexible region were not fully addressed. Shortening the linker connecting the TM domain to the cytosolic region likely restricts the amplitude of cytoplasmic domain motion and/or refolding, potentially obscuring physiologically relevant conformational transitions. Consistent with this notion, none of the reported cryo-EM structures show evidence of a two-helix bundle (THB), a fold previously identified by solution NMR in the canine NCX1 AD splice variant (residues 285–301 and 307–323; PDB ID: 6BV7) (Yuan et al., 2018). Instead, the homologous region in cardiac NCX1 - referred to as the exchanger inhibitory peptide (XIP) - adopts a conformation with two antiparallel β-strands in all cryo-EM models. At low Ca²⁺ concentrations, this β-structured XIP region interacts with CH2 and a β-hairpin, stabilizing the inactivated state of the transporter. A similar XIP configuration is also observed in the presence of the specific inhibitor SEA0400 (Dong et al., 2024). According to the mechanistic model proposed by Gao et al. (Xue et al., 2023), elevated cytosolic Ca²⁺ induces conformational changes centered on CBD2, resulting in a collision between the CH2 helix and the β-hub. This interaction destabilizes the β-hub and triggers detachment of the entire cytosolic domain from the TM module. The authors further speculate that this detachment could disengage the XIP region from the membrane and relieve constraints on TM1 and TM6, yielding a constitutively active TM domain. Under such conditions, it is conceivable that the XIP region may adopt an alternative fold, such as the THB observed by NMR, consistent with an “order-to-disorder-to-order” transition coupled to exchanger activation. Collectively, available evidence supports a model in which XIP acts as a dynamic, context-dependent regulatory element rather than a rigid structural motif. Its ability to engage in multiple interaction networks and to sample distinct conformational states positions XIP as a key mediator of NCX allostery, linking cytosolic ion sensing to TM-domain function.

Interestingly, Abiko et al. previously used solution NMR spectroscopy to demonstrate that Ca²⁺ binding modulates the interdomain flexibility between CBD1 and CBD2 in CALX, the Na⁺/Ca²⁺ exchanger from Drosophila melanogaster. This observation led to the proposal that the large intracellular loop behaves as an entropic spring, whose effective stiffness is controlled by Ca²⁺ binding to CBD1 and which, in turn, regulates ion transport by modulating the coupling between the cytosolic regulatory domain and the TM module. In this model, Ca²⁺-dependent changes in conformational entropy rather than large rigid-body rearrangements serve as the primary regulatory input (Abiko et al., 2016). The restrained flexibility was further conformed by Giladi et al. who utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments on full-size mammalian NCX1.4 variant to demonstrate that Ca²⁺ binding to CBD1 and CBD2 rigidifies the intracellular regulatory loop and promotes its interaction with the membrane domains (Giladi et al., 2024).

Further characterization of XIP dynamics and CBD1/CBD2 interactions in full-length, minimally constrained NCX constructs will be essential to fully understand how intrinsic disorder and conditional folding contribute to the remarkable regulatory versatility of this transporter family.

Pathogenic mutations linked to IDRs

In principle, mutations within intrinsically disordered regions might be expected to be relatively well tolerated, given the absence of stable tertiary structure. Nonetheless, multiple studies have linked IDR mutations in ion channels and transporters to human disease. In several cases, these variants likely act indirectly, by perturbing expression, stability, gating/activation, or assembly of the encoded integral membrane protein, rather than by disrupting a defined IDR-specific structural element.

For example, in TRPV4, variants within the intrinsically disordered portion of the N-terminal domain (NTD), including G20R, D62N, S93F, S94L, and P97R, have been associated with neuropathy/channelopathy phenotypes such as mild or recessive neuropathy, vocal cord weakness, and SMA (spinal muscular atrophy)-like presentation (Berth et al., 2025). In NaV1.5 (SCN5A), the Q1832E substitution within an intrinsically disordered inter-domain loop (IDL) produced clinical manifestations comparable to those observed in carriers of “damaging” variants with severe phenotypes, despite being predicted as “benign” by in silico tools (Tarantino et al., 2023). Finally, in ITPR1 (IP3 receptor 1), GLUT1 (SLC2A1) and CACNA1H (CaV3.2; T-type calcium channel), mutations in disordered cytosolic segments generate dileucine motifs that increase clathrin binding, consistent with a trafficking-based disease mechanism termed “dileucineopathies” (Meyer et al., 2018).

NMR experiments used to expose and characterize disordered regions

Solution NMR is ideally suited to describe the behavior of IDPs/IDRs because it provides ensemble-averaged structural and dynamic parameters that report on each assigned resonance in the molecule. NMR investigations begin by assigning spectral resonances to specific atoms; in proteins, the first step is obtaining backbone assignments. To accomplish this, methods based on ¹H/¹⁵N/¹³C triple-resonance experiments and multidimensional frequency separation were developed in the 1990s (Grzesiek and Bax, 1992; Yamasaki et al., 1994). Examples of the application of this approach to ion channel IDRs are discussed above (Bondarenko et al., 2022; Goretzki et al., 2022).

In practice, however, the high degree of disorder and flexibility in IDRs affects NMR spectra considerably. Although long coherence lifetimes associated with high flexibility provide the sensitivity needed to use sophisticated multidimensional pulse sequences, extensive motion leads to averaging effects that cause chemical-shift clustering, particularly in the proton dimension. Furthermore, rapid solvent exchange of amide protons at physiological pH, due to the unprotected nature of the IDR backbone, results in a loss of sensitivity in the ¹H/¹⁵N HSQC spectrum. This experiment is usually the first 2D spectrum examined to determine a target’s susceptibility to NMR investigation. Therefore, specialized techniques to circumvent the problems of severe overlap and exchange broadening of amide resonances with water have been introduced in recent years (Felli et al., 2021) and applied (Schiavina et al., 2024), mostly to soluble targets. Once assignments have been established, NMR can provide information about the presence of secondary structural elements within the region of interest, as shown for THB in NCX1 (Yuan et al., 2018), and about binding to potential targets through titration experiments, also known as chemical-shift mapping (as exemplified in (Goretzki et al., 2023; Goretzki et al., 2018; Li et al., 2010; Gayen et al., 2012)). The orientation between and within domains can be determined by measuring residual dipolar couplings (RDCs, as discussed in (Abiko et al., 2016)). Paramagnetic relaxation enhancement (PRE) measurements yield long-range distance restraints that are especially important for generating initial models during structure determination of domains with substantial conformational flexibility, as demonstrated in (Bondarenko et al., 2022).

Relaxation experiments spanning a wide range of time scales can provide dynamic information about IDRs and their complexes (as presented in (Bondarenko et al., 2022; Abiko et al., 2016)). Each type of relaxation measurement yields unique insight into biomolecular conformational changes and function, as highlighted in a concise yet comprehensive recent review (Figure 6) (Camacho-Zarco et al., 2022). NMR spin relaxation probes relaxation-active mechanisms on time scales ranging from tens of picoseconds to tens of nanoseconds, or even slower, and is typically governed by dipole–dipole interactions between neighboring nuclei that arise from macromolecular reorientation processes. ¹⁵N relaxation is usually the first approach explored when investigating protein dynamics. The canonical triad of ¹⁵N relaxation parameters comprises the ¹⁵N-¹H heteronuclear NOE and the longitudinal and transverse relaxation rates, R1 and R2, respectively. These parameters are essential for identifying fast backbone motions: they not only report the overall relaxation properties of the target but also reveal differences among regions that are directly linked to local mobility along the polypeptide chain. ¹⁵N-¹H NOE values are regarded as a particularly informative marker of flexibility, as they can become negative when extensive high-frequency (ps - ns) motions are present.

These same time scales are readily accessible to atomistic molecular dynamics (MD) simulations of fully solvated proteins, making the combination of MD and NMR especially powerful. In ensemble-based integrative modeling MD complements NMR by providing atomistic, time-resolved structural ensembles that can be validated and refined against NMR’s ensemble-averaged experimental observables (such as chemical shifts, RDC, PRE and relaxation measurement discussed above), therefore enabling mechanistic interpretation of dynamics, transient structure, and interactions, especially in disordered regions. Rather than yielding a single structure, the resulting model provides a weighted ensemble that quantifies contact probabilities, transient secondary-structure propensities, and population shifts upon binding, and can be cross-validated against independent restraint sets. Conversely, improved interpretation of NMR relaxation in terms of global and local molecular motions has driven advances in force-field accuracy, underscoring the synergy between these two atomic-resolution techniques (Showalter and Brüschweiler, 2007).

Conclusion

Intrinsically disordered regions are now understood to be active contributors to the operation of ion channels and transporters, not passive appendages. In many systems, these segments act as adaptable regulatory elements that translate cellular cues, such as binding events, partner recruitment, or post-translational modifications, into changes in gating, transport cycling, assembly, and trafficking. For integral membrane proteins, where functional transitions can involve multiple conformational routes and short-lived intermediates, disorder offers a practical solution: it enables conditional interactions and rapid redistribution of conformational populations without requiring a single rigid architecture.

Progress in linking IDR features to transport behavior increasingly depends on methods that can capture heterogeneity and dynamics directly. Solution NMR provides residue-level access to structural propensities, interaction surfaces, and motional time scales, while computational approaches, especially molecular dynamics and ensemble-based integrative modeling, translate these observations into atomistic, testable mechanisms. When used together, NMR and MD support quantitative descriptions of disordered ensembles and their reorganization upon membrane or targets binding, thereby bridging conformational behavior and physiological function.

At the same time, key obstacles persist. Disordered segments associated with membrane proteins remain challenging to interrogate experimentally because of resonance overlap, exchange effects, and the difficulty of maintaining native-like membrane environments. On the computational side, the accuracy of disordered-state sampling and protein-water/protein-lipid balance continues to depend on force-field performance and sufficient conformational exploration. These realities motivate integrative strategies that combine multiple orthogonal NMR readouts with ensemble-aware computational frameworks, allowing mechanistic conclusions to be drawn while explicitly accounting for uncertainty.

Future advances will likely come from tighter experimental-computational iteration performed under increasingly realistic membrane conditions (e.g., nanodiscs or other lipid-mimetic systems), coupled with improved labeling and long-range restraint strategies. A diverse array of existing and emerging tools, including circular dichroism, light scattering, fluorescence microscopy, steady-state and time-resolved fluorescence, Raman spectroscopy, in-cell NMR, and atomic force microscopy coupled with site-directed mutagenesis, should be integrated in future as well to provide a better understanding of how IDRs within ion channels and transporters regulate across-membrane transport. Such workflows should help discriminate between alternative models of regulation and clarify how cytosolic and/or extracellular disorder communicates allosterically with transmembrane cores. Ultimately, establishing a quantitative picture of IDR-driven control in transport proteins will deepen our understanding of membrane physiology and may expand therapeutic opportunities, since disordered segments frequently host regulatory sites, interaction hubs, and post-translational modification “switches” that can be targeted to modulate transport activity in disease-relevant pathways.

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Footnotes

Edited by: Frederic Harb, University of Balamand, Lebanon

Reviewed by: Abrar Hussain Qadri, University of Hyderabad, India

Imad Boulos, University of Balamand, Lebanon