Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

NMR structure of the transmembrane and cytoplasmic domains of human CD4 in micelles

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4⁺ cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes. Besides its cellular functions, CD4 serves as the main receptor for human immunodeficiency virus type I (HIV-1). During T-cell infection, the CD4 extracellular domain is bound by HIV-1 gp120, the viral surface glycoprotein, which triggers a number of conformational changes ultimately resulting in virion entry of the cell. Subsequently, CD4 is downregulated in infected cells by multiple strategies that involve direct interactions of the HIV-1 proteins VpU and Nef with the cytoplasmic part of CD4. In the present work, we describe the NOE-based solution structure of the transmembrane and cytoplasmic domains of the cystein-free variant of CD4 (CD4mut) in dodecylphosphocholine (DPC) micelles. Furthermore, we have characterized micelle-inserted CD4mut by paramagentic relaxation enhancement (PRE) agents and ¹H-¹⁵N heteronuclear NOE data. CD4mut features a stable and well-defined transmembrane helix from M372 to V395 buried in the micellar core and a cytoplasmic helix ranging from A404 to L413. Experimental data suggest the amphipathic cytoplasmic helix to be in close contact with the micellar surface. The role of the amphipathic helix and its interaction with the micellar surface is discussed with respect to the biological function of the full-length CD4 protein.     

Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits

In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (alpha2, alpha5, beta1, beta2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe(1129) in alpha2, to Phe(1026) in alpha5, to Ile(757) in beta1, and to His(728) in beta2. The alpha-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys(1125) in alpha2, Lys(1022) in alpha5, Lys(752) in beta1, and Lys(724) in beta2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed.     

Peptide probe study of the role of interaction between the cytoplasmic and transmembrane domains of the ryanodine receptor in the channel regulation mechanism

Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseases are localized to three easily definable regions: region 1 (N-terminal domain), region 2 (central domain), and a rather broad region 3 containing the channel pore. As shown in our recent studies, the interdomain interaction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here we present evidence that within region 3 there is a similar channel regulation mechanism mediated by an interdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significant activation of [3H]ryanodine binding above threshold Ca2+ concentrations (>or=0.3 microM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR1, and the site of cross-linking was identified by gel analysis of fluorescently labeled proteolytic fragments with the aid of Western blotting with site-specific antibodies. The shortest fluorescently labeled band was a 96 kDa fragment which was stained with an antibody directed to the region of residues 4114-4142 of RyR1, indicating that the interaction between the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containing the region of residues 4114-4142 is involved in the mechanism of Ca2+-dependent channel regulation. In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channel activation similar to that of DP15.     

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.     

Transmembrane domain-induced oligomerization is crucial for the functions of syndecan-2 and syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Calpha activation or in syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.     

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.     

Solution structure of the dimeric cytoplasmic domain of syndecan-4.

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.     

Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.     

Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single-spanning membrane protein. A 1H-NMR study

PMP1 is a 38-residue single-spanning membrane protein whose C-terminal cytoplasmic domain, Y25-F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H-nuclear magnetic resonance data of two synthetic fragments: F9-F38, i.e. 80% of the whole sequence, and Y25-F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25-F38 peptide adopts a well-defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non-native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.     

Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor

The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Here we report heterologous expression and purification of a CD4 fragment comprising the transmembrane and cytoplasmic domains of human CD4. A synthetic gene encoding CD4 amino acid residues 372-433 and a protease cleavage site was cloned into the pTKK19xb/ub plasmid. The CD4 fragment was expressed in Escherichia coli C43(DE3) cells as a ubiquitin fusion with an N-terminal His-tag, isolated, released by PreScission proteolytic cleavage, and purified to homogeneity. Incorporation of the recombinant CD4 fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. A high resolution NMR spectrum of uniformly (15)N-labeled CD4 peptide in membrane simulating micelles proves the possibility of solution NMR studies of this CD4 fragment and of its molecular complexes.     

A coiled-coil structure of the alphaIIbbeta3 integrin transmembrane and cytoplasmic domains in its resting state

One of the hallmark features of the integrin receptors is the ability to transmit signals bidirectionally through the cell membrane. The transmembrane integrin domains are pivotal to the signaling events. An understanding of the signaling mechanism requires structural information. Here, we report a structural model of the transmembrane and part of the cytosolic domains of the alphaIIbbeta3 integrin in its resting state. The model was obtained computationally by a restrained conformational search of helix-helix interactions. It agrees with one published NMR structure of the cytoplasmic complex and can put many experimental findings on structural grounds. According to our model, integrins form an intricately designed coiled-coil structure in the resting state. The conserved Glycophorin A (GpA)-like sequence motif of the alpha, but not the beta, subunit, is in the interface of this model. Based on our calculations and other data, a signaling mechanism that involves a transient GpA-like structure is proposed.     

Chirality-independent protein-protein recognition between transmembrane domains in vivo

Stereospecificity in protein-protein recognition and docking is an unchallenged dogma. Soluble proteins provide the main source of evidence for stereospecificity. In contrast, within the membrane little is known about the role of stereospecificity in the recognition process. Here, we have reassessed the stereospecificity of protein-protein recognition by testing whether it holds true for the well-defined glycophorin A (GPA) transmembrane domain in vivo. We found that the all-D amino acid GPA transmembrane domain and two all-D mutants specifically associated with an all-L GPA transmembrane domain, within the membrane milieu of Escherichia coli. Molecular dynamics techniques reveal a possible structural explanation to the observed interaction between all-D and all-L transmembrane domains. A very strong correlation was found between amino acid residues at the interface of both the all-L homodimer structure and the mixed L/D heterodimer structure, suggesting that the original interactions are conserved. The results suggest that GPA helix-helix recognition within the membrane is chirality-independent.     

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.     

Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.     

Clustering induces redistribution of syndecan-4 core protein into raft membrane domains

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, we have studied the distribution of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts as demonstrated by the sucrose gradient centrifugation and life confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. We conclude that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling.     

Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.