The unique structural features of transmembrane proteins include hydrophobic regions that embed within the membrane’s lipid core and hydrophilic regions exposed to aqueous surroundings. Combinations of transmembrane and extracellular regions allow transmembrane proteins to perform a range of functions, such as facilitating cell signaling and adhesion, maintaining cellular integrity, and transporting ions, nutrients, and waste products across the membrane.

Vital to the function of both prokaryotic and eukaryotic cells, transmembrane proteins regulate key processes like cellular communication, immune responses, and metabolic activity. Dysfunction or dysregulation of these proteins is associated with various diseases, including cystic fibrosis, autoimmune disorders, and several types of cancer.

A deeper understanding of transmembrane proteins is essential for advancing therapeutic approaches aimed at restoring their function and treating membrane-related disorders effectively.

Structure of transmembrane proteins

Transmembrane proteins traverse the lipid bilayer of cell membranes, with segments exposed to both extracellular and intracellular environments. These proteins are typically composed of α-helical regions containing 20 to 25 hydrophobic amino acids.

During synthesis, transmembrane proteins pass through the endoplasmic reticulum and Golgi apparatus, where they undergo glycosylation. This process links carbohydrates to the protein, resulting in glycoproteins with oligosaccharide chains exposed on the cell surface.

Hydrophobic and hydrophilic regions

A defining feature of transmembrane proteins is their amphipathic nature, meaning they possess both hydrophobic and hydrophilic regions. The hydrophobic segments are embedded within the lipid bilayer, interacting with the fatty acid tails of membrane lipids. In contrast, the hydrophilic regions are exposed to the aqueous environments of the cytoplasm and extracellular space, facilitating interactions with water-soluble molecules and ions.

Common structural motifs: alpha-helical and beta-barrel structures

Transmembrane proteins typically use alpha-helical or beta-barrel (closed beta-sheets forming toroidal structure) structural motifs. The alpha-helix is a common motif for single-pass transmembrane proteins, forming a helical structure across the membrane. In contrast, beta-barrels are typically found in proteins that span the membrane multiple times to create channels or pores, such as in the outer membranes of bacteria and mitochondria.

Types of transmembrane proteins

Transmembrane proteins can be classified based on their structure, orientation, and interaction with the lipid bilayer.

Single-pass (bitopic) and multi-pass (polytopic) proteins

Single-pass transmembrane proteins, also referred to as bitopic or single-spanning proteins, traverse the lipid bilayer only once. In contrast, multi-pass transmembrane proteins, characterized by two or more α-helical transmembrane domains, cross the plasma membrane multiple times. These multi-pass proteins often form intricate structures, such as ion channels and receptors.

For example, cadherins exemplify single-pass transmembrane proteins, while G-protein-coupled receptors (GPCRs) and bacteriorhodopsin serve as notable examples of multi-pass transmembrane proteins.

Integral membrane and peripheral membrane proteins

Membrane proteins are categorized into two main classes - integral and peripheral membrane proteins - based on their structure, function, and location within the membrane.

Integral membrane proteins

Integral membrane proteins are permanently embedded in the cell membrane, with portions of their structure extending into the lipid bilayer. Their hydrophobic surfaces enable integration into the membrane's hydrophobic core. These proteins may span the membrane completely (transmembrane proteins) or extend to only one side of the membrane (anchored proteins). Examples include transporters such as glucose transporters (sodium-glucose linked transporter-1 or SGLT1) ¹, channels such as the sodium-potassium pump ², receptors like the insulin receptor ³ and enzymes such as tyrosine kinase ⁴, which play important roles in cellular communication and substance transport.

Peripheral membrane proteins

Peripheral membrane proteins are loosely associated with the membrane's surface, primarily through electrostatic interactions. Unlike integral proteins, they can attach and detach from the membrane as needed. These proteins typically have hydrophilic residues on their surface and hydrophobic residues at their core. Peripheral proteins are either linked to integral membrane proteins (soluble proteins associated with membrane proteins) or part of the membrane skeleton, contributing to structural support. A few examples of peripheral membrane proteins include ankyrin ⁵, spectrin ⁶, and cytochrome c oxidase ⁷.

Both integral and peripheral membrane proteins are involved in essential processes such as molecular transport, the electron transport chain, and the expulsion of specific proteins from the cell. These functions are vital for maintaining cellular homeostasis and facilitating biological interactions.

Transmembrane proteins in different cellular membranes

• Plasma membrane: The transmembrane proteins of the plasma membrane allow for the transport of substances, signal transduction, and adhesion, among other processes. Their responsibilities are important for communicating with the extracellular environment, controlling cell signaling, mediating receptor-ligand interactions, and controlling ion and nutrient transport across the membrane.

• Organelle membranes: In organelles, transmembrane proteins have specialized functions. The majority of the mitochondrial membrane proteins play a vital role in oxidative phosphorylation and ATP synthesis, while ER membrane proteins are involved in protein synthesis, modification (eg, glycosylation), quality control, and calcium storage and signaling. Additionally, Golgi apparatus membrane proteins are vital for post-translational modifications, vesicle formation, and the sorting of proteins for intracellular trafficking.

• Specialized roles of transmembrane proteins in other specific structures: In specialized membranes, such as those of synaptic vesicles, transmembrane proteins facilitate the specific functions of the specific structure, such as in neurotransmitter release or ion homeostasis.

Folding mechanisms and transmembrane domains

Specialized molecular chaperones control the folding of transmembrane proteins, which occur co-translationally in the membrane. The transmembrane proteins are folded into precise three-dimensional structures as required for their activity, with a particular emphasis on producing hydrophobic domains that interact with the membrane. A few examples of molecular chaperones include heat shock protein 60 (Hsp 60) and Hsp 70⁸. The transmembrane domains (protein portions embedded in the membrane) of the protein are frequently rich in hydrophobic amino acids, which help to keep the protein stable within the membrane.

Membrane topology and interactions with the lipid bilayer

Certain lipid molecules (including the lipid composition of the cell membrane) act as covalent anchors, locking proteins within the lipid bilayer. These lipid anchors, consisting of prenyl and fatty acyl groups, embed in the membrane's hydrophobic core to stabilize protein-membrane connections, while glycosylphosphatidylinositol anchors ⁹ attach to the outer leaflet of the membrane. Hence, the lipid composition of cellular membranes helps in anchoring transmembrane proteins.

The nature of these lipid-protein interactions is important for correctly directing proteins to certain membrane areas, such as lipid rafts, and impacting protein structure. For example, when membrane proteins are reconstituted in membranes with differing lipid compositions, their orientation may change.

Impact of protein structure on membrane association

The folding pattern of transmembrane proteins can influence the efficiency of interaction with the cell membrane by ensuring proper orientation of hydrophobic and hydrophilic regions. For example, certain proteins may adopt conformations that necessitate extra molecular interactions, such as covalent lipid modifications (eg, palmitoylation, for example, of the endoplasmic reticulum thioredoxin-related transmembrane protein TMX¹⁰, myristoylation of cytochrome b reductase ¹¹, or prenylation of G protein-coupled receptors) ¹², enhance hydrophobicity, and facilitate anchoring to the lipid bilayer.

Furthermore, protein-protein interactions stabilize complexes to maintain their location in the membrane, as seen in the GX3G motif of glycophorin A (GpA) transmembrane helix where van der Waals forces and weak hydrogen bonding stabilize the association ¹³. Post-translational modifications (eg, glycosylation of CD98) ¹⁴ and chaperone-mediated folding, for example, a protein complex, PAT (protein associated with translocon) ¹⁵, a heterodimer of CCDC47 and Asterix play essential roles in maintaining proper protein structure and function within the membrane.

Functions of transmembrane proteins in cellular processes

Transmembrane proteins play a vital role in maintaining cellular function and facilitating communication between the cell and its environment. Their strategic positioning across the lipid bilayer enables them to perform several essential tasks.

Transport functions

Transport is one of the key roles of transmembrane proteins. The transport of waste materials, nutrients, and ions across the cell membrane is their core function. Transmembrane proteins are folded to form pumps, transporters, or channels based on the type of molecule(s) they facilitate the movement of. The preservation of cellular homeostasis is also intricately dependent on the constant movement of substances across membranes, both between the intracellular and extracellular membranes, as well as across membranes bounding organelles, vesicles, and other structures.

Ion channels and carrier proteins

Ion channels are pore-forming membrane proteins that create a passage to allow the passive movement of specific ions across the plasma membrane. Ion channels specifically allow the passage of inorganic ions, such as Na+, K+, Ca2+, and Cl- down their electrochemical gradient ¹⁶. The opening and closing of ion channels are gated, triggered by a stimulus (gated), a voltage change (voltage-gated), or the binding of a ligand (ligand-gated).

For example, the bacterial K+ channel contains four transmembrane subunits forming a pore in the cell membrane. The two transmembrane helices of each subunit form a cone tilting outside for the exit of potassium ions; a selectivity filter is formed by the pore helix (connecting these two helices) and a loop. This selectivity filter from four subunits forms a pore through which the ions are moved.

Carrier proteins, also known as transporters or permeases, facilitate the transport of molecules such as glucose or amino acids, either passively or actively. An example is the transmembrane ATPase Na+ K+ pump that maintains the osmotic equilibrium and membrane potential in cells for many functions, such as kidney reabsorption ¹⁷, brain action potentials, and motility of sperm. The pump undergoes alternation between the E1 state, where it has a high affinity for sodium and ATP; high-affinity Na binding sites are open to the cytoplasm, and the E2 state, where the enzyme has a high affinity for potassium and a low affinity for ATP.

The transition between states is accompanied by ADP release, the opening of the outer gate, and sodium ion release to the extracellular side. The pump has a catalytic alpha subunit and an auxiliary beta subunit; the alpha subunit has a transmembrane region with ten helices containing three binding sites that bind to Na+ in the E1 state and two binding sites that bind to K+ in the E2 state.

Active, passive, and facilitated diffusion mechanisms

• Passive diffusion occurs when molecules move through the membrane down their concentration gradient without the need for energy. An interesting example is the nuclear pore complexes or NPCs on the membrane of the nucleus ¹⁸. These NPCs are made of pore-forming proteins (nucleoporins) and allow the passive transport of smaller molecules (30-60kDa) ¹⁹.
• Facilitated diffusion uses carrier proteins to transport molecules more quickly through the membrane with no energy expenditure. A well-known example is the glucose transporter, which transports glucose and has 12 alpha-helical transmembrane segments ²⁰, such as the facilitated diffusion glucose transporters (GLUTs) like GLUT 2 on the liver, intestine, and kidney ²¹.
• Active transport requires energy, usually in the form of ATP, to transport molecules against their concentration gradient, essential for functions like nutrient uptake and waste removal. An example is the sodium-potassium pump that pumps out three sodium ions out of the cell and two potassium ions inside the cell with ATP hydrolysis for conformational changes of the protein pump shape for its function ²².

Signal transduction roles

Transmembrane signaling mechanisms involve integral membrane receptor proteins for recognizing and binding an external signal, as well as one or more effector proteins for generating intracellular signals.

Types of receptors

• G protein-coupled receptors (GPCRs) are involved in signaling pathways that transduce extracellular signals into intracellular signals that regulate physiological processes and control a variety of physiological systems, including vision, taste, and neurotransmission. GPCRs are the largest superfamily of membrane receptors with shared conserved seven transmembrane helices that are connected by three inter- and three intracellular loops ²³.

Ions, hormones, neurotransmitters, etc, stimulate allosteric signal transduction. Once activated, they produce secondary messengers using heterotrimeric G proteins (belonging to 4 families: (Gs, Gi/o, Gq/11, and G12/13) to initiate downstream pathways. The binding of a ligand to GPCRs causes its activation to catalyze the GDP/GTP exchange on the inactive G alpha subunit.

This results in the dissociation of G alpha from the G beta-gamma dimer. G alpha binds GTP, and the G alpha-GTP and G beta-gamma influence effector proteins, including phospholipase C, ion channels, and adenyl cyclase. These effector proteins produce second messengers like cyclic AMP (cAMP). The hydrolysis of GTP to GDP by the G alpha subunit causes the G alpha subunit to reassociate with G beta-gamma dimer and result in G protein inactivation.

• Enzyme-linked receptors, such as receptor tyrosine kinases (RTKs), perform important roles in cell development and differentiation, and in triggering enzymatic activity upon ligand binding. They act as signal receivers and enzymes and are essential for many signaling processes in the human body.

RTKs contain an extracellular ligand binding domain, a transmembrane helix, and an intracellular region (composed of a juxtamembrane regulatory region, a tyrosine kinase domain (TKD), and a carboxyl (C-) terminal tail) ²³. RTKs are activated by specific growth factors binding, leading to dimerization of the receptor (the active form), which causes autophosphorylation of the TKD. This autophosphorylation also recruits downstream signaling proteins with phosphotyrosine-binding (PTB) or Src homology-2 (SH2) domains to then activate downstream pathways, such as RAS/MAPK.

Cell adhesion and structural support

Transmembrane proteins play a vital role in cell adhesion, enabling cells to connect with one another or anchor to the extracellular matrix. Proteins such as cadherins mediate cell-cell adhesion, while integrins anchor cells to the extracellular matrix, providing structural stability. These interactions are essential for tissue integrity, development, and wound healing.

Enzymatic activity and implications for cellular processes

Some transmembrane proteins function as enzymes, catalyzing vital reactions at the cell membrane. For instance, adenylate cyclase converts ATP to cyclic AMP, a key signaling molecule. These enzymatic activities are integral to metabolic regulation, signal transduction pathways, and cellular responses to environmental changes.

Antigen presentation

In the immune system, transmembrane proteins such as major histocompatibility complex (MHC) molecules present antigens to immune cells. This process is vital for the activation of T-cells, allowing the immune system to recognize and respond to pathogens or infected cells. MHC molecules are classified into three families: MHC I on the surface of almost all nucleated cells that present any “abnormal” molecule, including viral particles or tumor antigens; MHC II on antigen-presenting cells like dendritic cells that activate immune responses; and MHC III is a tumor necrosis factor or a complement activator.

MHC I molecules contain two chains that are covalently bound to each other: a heavy chain (alpha chain) and a light chain (beta chain or beta2-microglobulin). The alpha1 and alpha2 domains of the alpha chain form a closed antigen-binding groove, the alpha3 domain is an extracellular domain, and the beta2 microglobulin stabilizes the structure²⁴. The MHC II molecules are made up of an alpha chain (containing alpha1 and alpha2 structural domains) and a beta chain (containing beta1 and beta2 structural domains); the alpha1 and beta1 domains form an open antigen-binding groove.

Functions in the nerve cell to facilitate the release and uptake of neurotransmitters

In nerve cells, transmembrane proteins are essential for synaptic communication. Proteins such as voltage-gated ion channels and neurotransmitter transporters regulate the release and reuptake of neurotransmitters, respectively, ensuring efficient signal transmission across synapses.

Voltage-gated ion channels are vital for an action potential where a stimulus causes the opening of voltage-gated Na+ channels (with automatic inactivation), allowing a small amount of Na+ to enter the cell down its electrochemical gradient ¹⁶. This influx of Na+ causes depolarization of the cell membrane, resulting in conformational rearrangements and the opening of voltage-gated K+ channels to allow the efflux of potassium to return the cell to its membrane potential ²⁵.

Neurotransmitter-gated ion channels are present in the central/peripheral nervous system on presynaptic and postsynaptic sites ²⁶. A presynaptic action potential causes the binding of neurotransmitter molecules to receptors to open the channel for ions to pass, allowing the quick transfer of information between neurons. Further, the dissociation of the neurotransmitter molecule from its receptor causes the closing of the channels.

This can be seen in the release of the amino acid glutamate within synaptic vesicles, wherein glutamate diffuses across the synaptic cleft and activates ionotropic glutamate receptors (iGluRs with an ion channel formed by the transmembrane domain) on the postsynaptic membrane to cause membrane depolarization.

Besides the direct synaptic mechanism, other modes of inactivation of ligand-activated channels include:

• Desensitization, a form of temporary deactivation, involves prolonged exposure to the ligand, which potentially renders the channel to become less sensitive, even if the ligand remains present.
• Phosphorylation, which involves specific enzyme-catalyzed addition of phosphate groups to the channel, changing its activity and causing inactivation.
• Receptor internalization, in which the channel is withdrawn from the cell membrane and internalized within the cell, resulting in decreased activity.
• Competitive inhibition, which occurs when other molecules attach to the receptor site, preventing the ligand from activating the channel.

Mechanisms of action for transmembrane proteins

Transmembrane proteins execute their functions through a range of mechanisms, leveraging their structural and biochemical properties to interact with the cell membrane, extracellular environment, and intracellular components.

Biogenesis and insertion into the membrane

Proper biogenesis and post-translational modifications are required for different activities of transmembrane protein, such as signal transduction, transport, and cell communication.

Protein synthesis, targeting, and membrane insertion mechanisms: Transmembrane proteins are synthesized in the cytoplasm and then transported to the membrane, where they are correctly inserted and folded. The process begins with protein synthesis on ribosomes, which are frequently associated with the endoplasmic reticulum (ER). Signal sequences on the nascent protein guide it to the ER membrane, where translocons facilitate insertion into the lipid bilayer.

Post-translational modifications: Once inserted in the lipid bilayer, the protein undergoes a variety of post-translational changes, including phosphorylation, glycosylation, and lipidation, all of which are vital for its stability, function, and membrane anchoring. These changes are also important for protein folding and interactions with other biological components.

Conformational changes and interactions with other molecules

Transmembrane proteins frequently undergo conformational changes that allow them to interact with and transport a variety of substances, including ligands, ions, and other proteins. Energy is required for active transport mechanisms to induce such conformational changes that allow molecules to move across membranes, such as in ATP-driven pumps (eg, Na⁺/K⁺-ATPase) or ion channels.

The Na⁺/K⁺-ATPase has been termed as the founding member of P-type ATPases ²⁷, using the energy from ATP hydrolysis for transporting the ions ²⁸. P-type ATPases have three cytosolic domains (A, P and N) and a membrane-embedded domain (M) containing six transmembrane helices; there are additional transmembrane helices for support ²⁹. They are auto-phosphorylated during catalysis at a conserved aspartate residue during the transport, where the phosphorylation reaction is carried out by the nucleotide-binding (N) domain and the A domain carries out the dephosphorylation.

This phosphorylation and dephosphorylation of the pump cause conformational changes in the membrane domain (M) in which the molecule to be transported is present. P-type ATPases alternate between the E1 conformation where high-affinity ion binding sites of Na+ (low affinity for the counter-transported ion K+) are exposed to the cytosol. The phosphorylation triggered by the ligand causes the shift to the E2 conformation where the ion binding sites are exposed to the outer side (these sites have high affinity for K+ and low affinity for Na+), causing an exchange.

Regulatory mechanisms and degradation of transmembrane proteins

Mechanisms like phosphorylation, ligand binding, and membrane composition change closely regulate the activity of transmembrane proteins. Damaged mitochondrial proteins are sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol through a process known as mitochondrial-associated degradation.

Correctly folded and functional transmembrane proteins that have fulfilled their role are often removed through controlled pathways in order to preserve cellular equilibrium. Usually ubiquitin-tagged, these proteins are either transported to lysosomes by endocytosis or autophagy, or they are directed to the ubiquitin-proteasome system for degradation.

Research has shown that proteins such as Get1/2, Sec61, and the ER membrane-protein complex (EMC) function in transmembrane domain insertion ³⁰. The endoplasmic reticulum (ER) quality control machinery recognizes correctly folded proteins to be trafficked to their final destinations. The misfolded transmembrane domains in the membrane section of a membrane protein are recognized and removed from the lipid bilayer degradation in the proteasome. The factors involved in this function have been identified by early research to be Hrd1 (recognizes the transmembrane domains and forms a retro-translocation channel) and Derlin ³⁰.

Removal and recycling of transmembrane proteins

Transmembrane proteins are eliminated from the membrane through endocytosis. Proteins, once internalized, can either be destroyed or recycled back to the membrane, depending on the cellular demands and the protein’s condition. This dynamic process protects the stability of membrane proteins and cellular function.

Correctly folded and functioning proteins, having served their purpose, are recycled via endosomal sorting and trafficking mechanisms. The retromer complex (Vps35, Vps29, Vps26) and Rab GTPases (Rab4, Rab11) regulate their transport and recycling to the plasma membrane or trans-Golgi network ³¹. Endosomal recycling minimizes wasteful degradation and maintains protein homeostasis. Proteins that are not recycled are sent to lysosomes for breakdown using the endosomal sorting complexes needed for the transport mechanism. This dynamic mechanism maintains cellular functionality and resource efficiency.

Importance of transmembrane proteins in health and disease

Transmembrane proteins help maintain cellular and physiological homeostasis, and thus influencing health. However, their aberrant functioning or expression contributes to the pathogenesis of various diseases when disrupted. Their roles in processes such as molecular transport, signal transduction, and cell adhesion highlight their importance in both physiological and pathophysiological states.

Role in maintaining cellular health and integrity

Transmembrane proteins are essential for cellular health and integrity as they control nutrient intake, ion balance, signal transduction, and cellular communication. Disruptions in their function can lead to health consequences, such as cancer, neurological disorders, and metabolic issues.

Implications of malfunction in transmembrane proteins

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, leading to defective Cl- transport across membranes. Transmembrane proteins have been implicated in autoimmune diseases such as multiple sclerosis, where there are reports of increased transmembrane protein 106B (TMEM106B) expression in multiple sclerosis plaques ^32,33. Similarly, neurological illnesses like epilepsy and neuropathic pain are caused by ion channel malfunction, which includes voltage-gated Na+ and K+ channels.

There are nine sub-types of the alpha sub-types of voltage-gated sodium channels (NaV1.1-NaV1.9). Mutations in NaV1.1 (SCN1A), NaV1.2 (SCN2A), NaV1.3 (SCN3A), NaV1.6 (SCN8A) and NaV1.7 (SCN9A) have been associated with epilepsy ³⁴. The most frequently mutated gene in genetic epilepsies is the SCN1A gene encoding the α subunit NaV1.1, with 1528 mutations described for epileptic diseases ³⁴.

In an interesting study, the use of gene therapy with antisense oligonucleotides against SCN1A in mice and non-human primates ³⁵. Several patients (28.6%) with idiopathic small nerve fiber neuropathy have mutations in voltage-gated sodium channel Na (V)1.7 ³⁶. Close to 10% of around 80 potassium channels have been linked to epilepsies and associated phenotypes, for example, Kv7.2 or Kv7.3 channels in neonatal epilepsy (benign familial nocturnal convulsions) and Kv1.2 channel genes in juvenile epilepsy ³⁷.

Drug targets that have been explored in research include the anti-epileptic drug ezogabine (EZG), which shifts the availability of Kv7.2 and Kv7.3 channels to more hyperpolarized potentials. A study on diabetic peripheral neuropathy patients reported that 70% of pathogenic variants were in SCN7A, SCN9A, SCN10A and SCN11A, where the frequencies of SCN9A and SCN11A variants were the highest in painful- small fiber neuropathy patients ³⁸. Diabetic mice and type 2 diabetes patients showed a reduction in the juxtaparanodal Kv1.2-subunits (potassium channels) ³⁹.

These studies outline the involvement of voltage channels in epilepsy and neuropathic pain; research is identifying drugs, such as the Nav1.7 blocker PF-05089771, the Nav1.8 blocker VX-548, and the potassium channel Kv7.2/7.3/7.5 opener ICA-105665 to target these altered channels/functions ⁴⁰. Overexpression or mutations in transmembrane proteins such as EGFR and receptor tyrosine kinases encourage uncontrolled cell proliferation and survival, which contributes to tumor formation.

Transmembrane proteins as therapeutic targets

Transmembrane proteins are prime targets for drug development. GPCRs, ion channels, and transporters are key drug targets in numerous diseases ranging from cancer to neurological disorders.

Below is a table of diseases commonly associated with the different types of transmembrane proteins and the drugs used to target them:

proteins and the drugs used to target them:

Additionally, research continues to identify various transmembrane proteins as the cause of several diseases. Highlights of recent findings are given below.

• GPCRs: Disease-causing mutations have been documented in 55 GPCRs for at least 66 monogenic human diseases ⁴¹. For example, variants of Group C GPCRs calcium-sensing receptor (CaSR) have been associated with mineral ion pathologies: inactivation causes hypocalciuric hypercalcemia type 1 and neonatal severe hyperparathyroidism (NSHPT); activating variants are associated with Bartter’s syndrome type V and autosomal dominant hypocalcemia type 1. Studies have shown the initial promise of calcilytics, such as NPSP795, in treating autosomal dominant hypocalcemia type 1.
  Research has also highlighted the role of G protein-coupled receptor kinases (GRKs) in cancer, such as high GRK2 levels and reduced GRK5 levels that influence thyroid-stimulating hormone receptor (TSHR) in differentiated thyroid carcinoma ⁴². GRK2 was also identified to be involved in skin cancer, gastric cancer, and tumor vessel formation. However, GRK inhibitors are yet to be brought into the clinic.

• Ion channels: Several studies suggest that regulating the function of key ion channels involved in diseases can effectively alleviate the symptoms, and for treating neurological disorders ⁴³.

The low-voltage activated T-type calcium channel (Cav3.2) has a low threshold for action potential firing in dorsal root ganglion neurons and plays a key role in treating peripheral pain. Another study showed that the T-type calcium channel blocker (KTt-45) could suppress growth and induce cell death in HeLa cells ⁴⁴. The transient receptor potential channels (TRP channels), the sensors for perception, have helped researchers understand the molecular basis of hearing with the potential for treating hearing loss.

The potassium channel Kv1.1 (KCNA1) has been associated with and was overexpressed in medulloblastoma and cervical cancer ⁴⁵; Kv1.3 (KCNA3) was overexpressed in cancers of the lung, colon, prostate, breast, pancreas, skeletal muscle, smooth muscle, melanoma, and lymph node cancers. A study reported a monoclonal antibody (Y4) developed against KCNK9, a member of the two-pore domain potassium (K2P) channels that could suppress human lung cancer xenografts and breast cancer metastasis in mice ⁴⁶.

• Transporters: The solute carrier (SLC) sub-groups of membrane proteins include more than 400 solute carriers with 65 families in humans⁴⁷. An example of their association with diseases is riboflavin transporters (RFVT, SLC52) with an autosomal recessive disorder known as multiple acyl-CoA dehydrogenase deficiency (MADD)⁴⁸. The neurodegenerative disease amyotrophic lateral sclerosis (ALS) is caused by increased levels of glutamate that cause degeneration of neurons. ALS is associated with the loss of excitatory amino acid transporter 1 (EAAT1) or SLC1A3, a glutamate transporter that belongs to the solute carrier 1 (SLC1) family of transporters.

The drug used to treat ALS, riluzole, inhibits postsynaptic neuron activation, prevents the release of glutamate and also lowers the levels of glutamate in the synaptic cleft to increase SLC1A3 activity. Developmental and epileptic encephalopathy (DEE) type 41, a treatment-refractory, was also associated with SLC1A2 (encodes sodium-dependent glutamate transporter 2 or EAAT2 that reduces the glutamate levels to prevent overstimulation and cell death) variants. Research has also reported promising results for DEE41 with valproic acid, recombinant interleukin 1 receptor antagonist (anakinra), and a pyrazine derivative - LDN/OSU-0212320.

• Adhesion molecules: Connexins are transmembrane proteins on the plasma membrane surface, of which many human cells abundantly express the gap-junctional protein connexin 43 (Cx43) ⁴⁹. Given the role of this protein in wound healing, the efficacy and safety of a peptide mimetic of the C-terminus of Cx43 (ACT1) chronic diabetic foot ulcers (DFUs) were assessed in a prospective, randomized, multicenter clinical trial ⁵⁰. Patients who received the topical application of ACT1 had a more significant reduction in ulcer area (72.1%) vs. those without it (57.1%); more patients reached 100% reepithelization of the ulcer and ACT1 was not immunogenic and with no serious adverse effects.

In a study on the cell adhesion molecule receptor integrin, mesenchymal drug-resistant cells express integrin beta-3 to potentially overcome chemoresistance; atorvastatin was identified as a novel drug candidate by in silico analysis that could sensitize cancer cells to chemotherapeutic drugs, showing its application to address drug resistance ⁵¹.

Methods for studying transmembrane proteins

Transmembrane proteins are challenging to study due to their amphipathic nature and integration into lipid bilayers. However, advances in biophysical, biochemical, and computational techniques have made it possible to unravel their structures and functions.

Techniques for structural analysis

Advanced techniques like X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM) are frequently used to study the structure of transmembrane proteins.

• X-ray crystallography produces high-resolution atomic-level features, but it requires the protein to form crystals, which is often difficult for membrane proteins. For example, X-ray crystal structures have been characterized for chemokine receptors (GPCR family with 7 transmembrane helices) that play a vital role in leukocyte migration and survival ⁵². Examples of such structures include CRCX4 (the first chemokine receptor identified as HIV-1 co-receptor), CCR2 (implicated in neurodegenerative diseases), and CCR9 (a target being studied for inflammatory bowel disease).

• NMR can provide insights into protein dynamics in solutions, although it is limited by protein size. NMR has been used to study the structures of several transmembrane proteins, such as the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA) and the seven helix-GPCR chemokine receptor CXCR1⁵³. The first protein to be structurally characterized by solution NMR in micelles was the phototaxis receptor sensory rhodopsin II (pSRII) ⁵⁴ that functions in blue-light avoidance in Natronomonas pharaonis. Solid state NMR was used to study the second transmembrane segment, M2, of the acetylcholine receptor and found that it formed a funnel-like channel by rotating its transmembrane alpha-helix ⁵⁵.

• Cryo-EM has emerged as a potent method for visualizing membrane proteins in their native states without the need for crystallization. The technique involves the rapid freezing of samples and trapping them in ice by freezing at below -150°C followed by imaging the sample with electron microscopy ⁵⁶. In one study, cryo-EM was used to visualize the folding process of how the beta-barrel assembly machine (BAM) folds transmembrane beta-barrel proteins into the outer membrane of Gram-negative bacteria ⁵⁷.

Functional assays and computational modeling

• Functional assays are used to determine the activities of transmembrane proteins, such as transport and receptor binding.

• Computational modeling enhances experimental procedures by simulating protein structures and interactions, predicting functional locations, and investigating conformational changes in silico.

Challenges in transmembrane protein research

• Difficulties in purification: Transmembrane proteins are difficult to purify due to their hydrophobic domains, making them difficult to isolate while retaining their structure. These proteins frequently require specialized detergents or lipid environments for optimum folding and stability.

• Maintaining stability and functional integrity: Another key problem is ensuring the stability and functional integrity of transmembrane proteins outside of the cellular membrane, as they are prone to denaturation in non-native settings. Specialized techniques and membrane-mimicking systems, such as nanodiscs or amphipols, are frequently used to address this concern.

Recent advancements in technologies for structural analysis

Recent advances in cryo-EM and membrane protein expression technologies have significantly increased the resolution and accessibility of structural research. New computational methods and advancements in in vitro lipid bilayer systems improve the ability to investigate these complex proteins, pushing the boundaries of membrane protein research.

Future directions and innovations in transmembrane protein research

Ongoing research on transmembrane proteins is focused on overcoming challenges related to their expression, purification, and structural analysis. Advances in high-throughput screening, synthetic biology, and structural techniques such as cryo-EM have enhanced our understanding of protein dynamics, signaling, and transport functions. Researchers are now investigating the roles of membrane proteins in disease mechanisms, with a particular focus on cancer, neurodegenerative disorders, and metabolic diseases.

Emerging therapeutic applications and biotechnological uses

Emerging therapeutic applications focus on targeting membrane proteins for drug development. GPCRs, ion channels, and transporters are key targets for novel therapies, including small-molecule drugs, biologics, and gene therapies. Many transmembrane proteins are being studied as targets of protein inhibitors for cancer treatment, such as c-Met (hepatocyte growth factor receptor or tyrosine-protein kinase MET) ⁵⁸.

After long-lasting results in patients with brain metastases in the GEOMETRY mono-1 trial, the FDA, in 2020, approved a selective, oral c-Met inhibitor molecule with high selectivity beneficial for patients with non-small cell lung cancer (NSCLC) having MET exon 14 skipping mutations. Monoclonal antibodies targeting calcitonin gene-related peptide (CGRP)⁵⁹, a member of the GPCR family, such as Erenumab and Eptinezumab have been approved by the FDA for treating/preventing migraine ⁶⁰.

For gene therapy, a study developed engineered prokaryotic sodium channels that were driven by muscle-specific promoters with the aim of treating cardiac arrhythmia ⁶¹. These engineered channels could lower conduction block and reentrant arrhythmias in fibrotic cardiac cultures and increase excitability and conduction in in vitro rats and human cardiomyocytes, paving the way for further research on gene therapy using engineered sodium channels.

The non-functional variant of the ATP-sensing pore-forming ion channel P2X7, also known as nfP2X7, is upregulated in several cancers ⁶². A phase I study for BIL010t, a polyclonal antibody targeting nfP2X7 for treating basal cell carcinoma, reported that the topical application of this antibody to lesions was safe and well-tolerated ⁶³. Advances in gene editing technologies like CRISPR and membrane protein engineering also hold promise for treating genetic disorders caused by defective membrane proteins.

Potential impact of transmembrane protein research on disease treatment

The impact of transmembrane protein research on disease treatment is substantial. Therapies are aimed at correcting misfolded proteins, restoring proper function, and improving targeted drug delivery to potentially revolutionize treatments for conditions like cystic fibrosis, cancer, and neurological diseases.

FAQs

What are transmembrane proteins?

Transmembrane proteins are a type of integral protein that traverses the lipid bilayer (the primary structural component of biological membranes). These proteins have an extraordinary ability to transcend the membrane, allowing them to interact with both the inner and outside surroundings of cells. They serve as an important component in many cellular functions by allowing communication, transporting chemicals, and mediating interactions between the cell and its surroundings.

How do transmembrane proteins contribute to cell signaling?

Transmembrane proteins promote cell signaling by functioning as receptors for external signals such as hormones or neurotransmitters. When ligands bind, the transmembrane protein receptors undergo conformational changes, activating intracellular signaling cascades that regulate cellular activities such as growth, differentiation, and metabolism, thereby allowing the cell to adapt to its surroundings.

How do transmembrane proteins maintain cellular integrity?

Transmembrane proteins maintain cellular integrity by controlling selective transport, cell signaling, and structural stability. They regulate ion balance, nutrition exchange, and waste elimination while connecting the cytoskeleton to the extracellular matrix. Furthermore, they aid in cell-to-cell communication, immunological responses, and intracellular trafficking, guaranteeing appropriate cellular function and homeostasis.

Why are transmembrane proteins important in disease?

Transmembrane proteins are important in disease because they influence essential physiological processes such as signaling, transport, and cell adhesion. Mutations or malfunctions in these proteins can alter normal cellular activities, resulting in cancer, neurological diseases, cardiovascular disorders, and inherited diseases like cystic fibrosis.

References

1. Navale, A.M., Paranjape, A.N. Glucose transporters: physiological and pathological roles. Biophysical reviews. 8, 5-9 (2016).
2. Pirahanchi, Y., Jessu, R., Aeddula, N.R. Physiology, sodium potassium pump. [Updated 2023 Mar 13]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537088/
3. Qingxin, L., Ying, L.W., CongBao, K. Solution structure of the transmembrane domain of the insulin receptor in detergent micelles. Biochimica et biophysica acta – Biomembranes. 1838, 1313-1321 (2014).
4. Li, E., Hristova, K. Receptor tyrosine kinase transmembrane domains: function, dimer structure and dimerization energetics. Cell adhesion & migration. 4, 249-254 (2010).
5. Staufenbiel, M., Lazarides, E. Ankyrin is fatty acid acylated in erythrocytes. Proceedings of the national academy of sciences of the United States of America. 83, 318-322 (1986).
6. Bonetta, L. Spectrin is peripheral.Journal of cell biology. 170, 12-13 (2005).
7. Garrido, C., Galluzzi, L., Brunet, M., et al. Mechanisms of cytochrome c release from mitochondria. Cell death & differentiation. 13, 1423-1433 (2006).
8. Saibil, H. Chaperone machines for protein folding, unfolding and disaggregation. Nature reviews molecular cell biology. 14, 630-642 (2013).
9. Paulick, M.G., Bertozzi, C.R. The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry. 47, 6991-7000 (2008).
10. Sanja B., Mathieu B.F. Gisou van der Goot. What does S-palmitoylation do to membrane proteins? The FEBS journal. 280, 2766-2774, (2013).
11. Shen, J., Wu, G., Tsai, AL., et al. Transmembrane helices mediate the formation of a stable ternary complex of b5R, cyt b5, and SCD1. Communications biology. 5, 956 (2022).
12. Arnold D.L., Liliane S. Flip-flopping retinal in microbial rhodopsins as a template for a farnesyl/prenyl flip-flop model in eukaryote GPCRs. Frontiers in neuroscience. 13, 2019.
13. Moore, D.T., Berger, B.W., DeGrado, W.F. Protein-protein interactions in the membrane: sequence, structural, and biological motifs. Structure. 16, 991-1001 (2008).
14. Console, L., Scalise, M., Salerno, S. et al. N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98). Scientific reports. 12, 14570 (2022).
15. Smalinskaitė, L., Kim, M.K., Lewis, A.J.O., et al. Mechanism of an intramembrane chaperone for multipass membrane proteins. Nature. 611, 161-166 (2022).
16. Alberts, B., Johnson, A., Lewis, J., et al. Molecular biology of the cell. 4th edition. New York: Garland Science; 2002. Ion Channels and the Electrical Properties of Membranes. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26910/
17. Pirahanchi, Y., Jessu, R., Aeddula, N.R. Physiology, sodium potassium pump. [Updated 2023 Mar 13]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537088/
18. Bindra, D., Mishra, R.K. In Pursuit of distinctiveness: transmembrane nucleoporins and their disease associations. Frontiers in oncology. 11, 784319 (2021).
19. Benjamin L.T., Barak R., Roxana M., et al. Simple rules for passive diffusion through the nuclear pore complex. Journal of cell biology. 215, 57-76 (2016).
20. Cooper, G.M. The cell: a molecular approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Transport of Small Molecules. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9847/
21. Sun, B., Chen, H., Xue, J. et al. The role of GLUT2 in glucose metabolism in multiple organs and tissues. Molecular biology reports. 50, 6963–6974 (2023).
22. Chen, I., Lui, F. Physiology, active transport. [Updated 2022 Sep 12]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK547718/
23. Zhang, M., Chen, T., Lu, X., et al. G protein-coupled receptors (GPCRs): advances in structures, mechanisms, and drug discovery. Signal transduction and targeted therapy. 9, 88 (2024).
24. Wu, Y., Zhang, N., Hashimoto, K., et al. Structural comparison between MHC classes I and II; in evolution, a class-II-like molecule probably came first. Frontiers in immunology. 12, 621153 (2021).
25. Grizel, A.V., Glukhov, G.S., Sokolova, O.S. Mechanisms of activation of voltage-gated potassium channels. Acta naturae. 6, 10-26 (2014).
26. Smart, T.G., Paoletti, P. Synaptic neurotransmitter-gated receptors. Cold Spring Harbor perspectives in biology. 4, a009662 (2012).
27. Clausen, M.V., Hilbers, F, Poulsen H. The structure and function of the Na,K-ATPase isoforms in health and disease. Frontiers in physiology. 8, 371 (2017).
28. Kühlbrandt, W. Biology, structure and mechanism of P-type ATPases. Nature reviews molecular cell biology. 5, 282–295 (2004).
29. Palmgren, M. P-type ATPases: many more enigmas left to solve. Journal of biological chemistry. 299, 105352 (2023).
30. Alina, G., Ramanujan, S.H. Transmembrane domain recognition during membrane protein biogenesis and quality control. Current biology. 28, R498-R511 (2018).
31. Vagnozzi, A.N., Praticò, D. Endosomal sorting and trafficking, the retromer complex and neurodegeneration. Molecular psychiatry. 24, 857-868 (2019).
32. TMEM39A transmembrane protein 39A [ Homo sapiens (human)]. Available on: https://www.ncbi.nlm.nih.gov/gene/55254
33. Bridget, S.-Z., Simone, S., James, E.G., et al. TMEM106B is increased in multiple sclerosis plaques, and deletion causes accumulation of lipid after demyelination. BioRxiv.  05.490697 (2022).
34. Menezes, L.F.S, Sabiá, J.E.F., Tibery, D.V., et al. Epilepsy-related voltage-gated sodium channelopathies: a review. Frontiers in pharmacology. 11, 1276 (2020).
35. Hsiao, J., Yuan, T.Y., Tsai, M.S., et al. Upregulation of haploinsufficient gene expression in the brain by targeting a long non-coding RNA improves seizure phenotype in a model of Dravet syndrome. EBioMedicine. 9, 257-277 (2016).
36. Faber, C.G., Hoeijmakers, J.G., Ahn, H.S., et al. Gain of function Naν1.7 mutations in idiopathic small fiber neuropathy. Annals of neurology. 71, 26-39 (2012).
37. Köhling, R., Wolfart, J. Potassium channels in epilepsy. Cold Spring Harbor perspectives in medicine. 6, a022871 (2016).
38. Almomani, R., Sopacua, M., Marchi, M., et al. Genetic Profiling of Sodium Channels in Diabetic Painful and Painless and Idiopathic Painful and Painless Neuropathies. International journal of molecular science. 24, 8278 (2023).
39. Zenker, J., Poirot, O., de Preux Charles, A.S., et al. Altered distribution of juxtaparanodal kv1.2 subunits mediates peripheral nerve hyperexcitability in type 2 diabetes mellitus. Journal of neuroscience. 32, 7493-7498 (2012).
40. Qi, W., Yifei, Y., Linghui, Y. Painful diabetic neuropathy: the role of ion channels. Biomedicine & pharmacotherapy. 173, 116417 (2024).
41. Thompson, M.D., Percy, M.E., Cole, D.E.C., et al. (2024). G protein-coupled receptor (GPCR) gene variants and human genetic disease. Critical reviews in clinical laboratory sciences. 61, 317–346 (2024).
42. Yu, S., Sun, L., Jiao, Y., et al. The role of G protein-coupled receptor kinases in cancer. International journal of biological sciences. 14, 189-203 (2018).
43. Hou, P., Du, X., An, H. Editorial: Ion channels: therapeutic targets for neurological disease. Frontiers in molecular neuroscience. 14, 797327 (2021).
44. Du, N.H., Ngoc, T.T.B., Cang, H.Q., et al. KTt-45, a T-type calcium channel blocker, acts as an anticancer agent by inducing apoptosis on HeLa cervical cancer cell line. Scientific reports. 13, 22092 (2023).
45. Zúñiga, L., Cayo, A., González, W., et al. Potassium channels as a target for cancer therapy: current perspectives. Onco targets and therapy. 15, 783-797 (2022).
46. Sun, H., Luo, L., Lal, B., et al. A monoclonal antibody against KCNK9 K (+) channel extracellular domain inhibits tumor growth and metastasis. Nature communication. 7, 10339 (2016).
47. Ayka, A., Şehirli, A.Ö. The role of the SLC transporters protein in neurodegenerative disorders. Clinical psychopharmacology and neuroscience. 18, 174-187 (2020).
48. Hediger, M.A., Clémençon, B., Burrier, R.E., et al. The ABCs of membrane transporters in health and disease (SLC series): introduction. Molecular aspects of medicine. 34, 95-107 (2013).
49. Xinhai, X., Wenjie, C., Cheng, C. Analysis of the function and therapeutic strategy of connexin 43 from its subcellular localization. Biochimie. 218, 1-7 (2024).
50. Grek, C.L., Prasad, G.M., Viswanathan, V., et al. Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: A multicenter, randomized trial. Wound repair and regeneration. 23, 203-212 (2015).
51. Janiszewska, M., Primi, M.C., Izard, T. Cell adhesion in cancer: beyond the migration of single cells. Journal of biological chemistry. 295, 2495-2505 (2020).
52. Arimont, M., Sun, S.L., Leurs, R., et al. Structural analysis of chemokine receptor-ligand interactions. Journal of medicinal chemistry. 60, 4735-4779 (2017).
53. Liang, B., Tamm, L.K. NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nature structure and molecular biology. 23, 468-474 (2016).
54. Marassi, F.M., Opella, S.J. NMR structural studies of membrane proteins. Current opinion in structural biology. 8, 640-648 (1998).
55. Marassi, F.M., Opella, S.J. NMR structural studies of membrane proteins. Current opinion in structural biology. 8, 640-648 (1998).
56. Cryo electron microscopy: principle, strengths, limitations and applications. https://www.technologynetworks.com/analysis/articles/cryo-electron-microscopy-principle-strengths-limitations-and-applications-377080
57. Doyle, M.T., Jimah, J.R., Dowdy, T., et al. Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Cell. 185, 1143-1156.e13 (2022).
58. Zhong, L., Li, Y., Xiong, L. et al. Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. Signal transduction and targeted therapy. 6, 201 (2021).
59. Moore, E.L., Salvatore, C.A. Targeting a family B GPCR/RAMP receptor complex: CGRP receptor antagonists and migraine. British journal of pharmacology. 166, 66-78 (2012).
60. Yuan, W., et al. RNA therapeutics in targeting G protein-coupled receptors: recent advances and challenges. Molecular therapy nucleic acids. 35, 102195 (2024).
61. Nguyen, H.X., Wu, T., Needs, D., et al. Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy. Nature communication. 13, 620 (2022).
62. Hutchings, C.J., Colussi, P., Clark, T.G. Ion channels as therapeutic antibody targets. Mabs. 11, 265-296 (2019).
63. Gilbert, S.M., Gidley B.A., Glazer, SJ.A., et al. A phase I clinical trial demonstrates that nfP2X7‐targeted antibodies provide a novel, safe and tolerable topical therapy for basal cell carcinoma, British journal of dermatology. 177, 117-124 (2017).