Mechanism and molecular determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by extracellular pH

The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa approximately 6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.     

Conformational changes of pore helix coupled to gating of TRPV5 by protons

The transient receptor potential channel TRPV5 constitutes the apical entry pathway for transepithelial Ca2+ transport. We showed that TRPV5 was inhibited by both physiological intra- and extracellular acid pH. Inhibition of TRPV5 by internal protons was enhanced by extracellular acidification. Similarly, inhibition by external protons was enhanced by intracellular acidification. Mutation of either an extra- or an intracellular pH sensor blunted the cross-inhibition by internal and external protons. Both internal and external protons regulated the selectivity filter gate. Using the substituted cysteine accessibility method, we found that intracellular acidification of TRPV5 caused a conformational change of the pore helix consistent with clockwise rotation along its long axis. Thus, rotation of pore helix caused by internal protons facilitates closing of TRPV5 by external protons. This regulation by protons likely contributes to pathogenesis of disturbances of Ca2+ transport in many diseased states. Rotation of pore helix may be a common mechanism for cross-regulation of ion channels by extra- and intracellular signals.     

TRPV1 acts as proton channel to induce acidification in nociceptive neurons

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca(2+)-dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na(+), Mg(2+), and Ca(2+). The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.     

Proton inhibition of unitary currents of vanilloid receptors

Protons, which are released during inflammation and injury, regulate many receptors and ion channels involved in pain transduction, including capsaicin channels (transient receptor potential vanilloid receptors 1). Whereas extracellular acidification both sensitizes and directly activates the channel, it also causes concomitant reduction of the unitary current amplitudes. Here, we investigate the mechanisms and molecular basis of this inhibitory effect of protons on channel conductance. Single-channel recordings showed that the unitary current amplitudes decreased with extracellular pH in a dose-dependent manner, consistent with a model in which protons bind to a site within the channel with an apparent pKa of ∼6. The inhibition was voltage dependent, ∼65% at −60 mV and 37% at +60 mV when pH was reduced from 7.4 to 5.5. The unitary current amplitudes reached saturation at [K⁺] ≥ 1 M, and notably the maximum amplitudes did not converge with different pHs, inconsistent with a blockade model based on surface charge screening or competitive inhibition of permeating ions. Mutagenesis experiments uncovered two acidic residues critical for proton inhibition, one located at the pore entrance and the other on the pore helix. Based on homology to the KcsA structure, the two acidic residues, along with another basic residue also on the pore helix, could form a triad interacting with each other through extensive hydrogen bonds and electrostatic contacts, suggesting that protons may mediate the interactions between the selectivity filter and pore helix, thereby altering the local structure in the filter region and consequently the conductance of the channel.     

Regulation of TRPV5 Single-Channel Activity by Intracellular pH

The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside–out patches of Chinese hamster ovary cells. The slope conductance values (Na⁺ as a charge carrier, between −25 and −75 mV) for full and subconductance opening at intracellular pH 7.4 were 59 ± 6 and 29 ± 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open–subconductance–closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance. Seung-Kuy Cha and Wasey Jabbar contributed equally to this work.     

Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels

Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.     

Mechanisms of proton inhibition and sensitization of the cation channel TRPV3

TRPV3 is a temperature-sensitive, nonselective cation channel expressed prominently in skin keratinocytes. TRPV3 plays important roles in hair morphogenesis and maintenance of epidermal barrier function. Gain-of-function mutations of TRPV3 have been found in both humans and rodents and are associated with hair loss, pruritus, and dermatitis. Here, we study the mechanisms of acid regulation of TRPV3 by using site-directed mutagenesis, fluorescent intracellular calcium measurement, and whole-cell patch-clamp recording techniques. We show that, whereas extracellular acid inhibits agonist-induced TRPV3 activation through an aspartate residue (D641) in the selectivity filter, intracellular protons sensitize the channel through cytoplasmic C-terminal glutamate and aspartate residues (E682, E689, and D727). Neutralization of the three C-terminal residues presensitizes the channel to agonist stimulation. Molecular dynamic simulations revealed that charge neutralization of the three C-terminal residues stabilized the sensitized channel conformation and enhanced the probability of α-helix formation in the linker between the S6 transmembrane segment and TRP domain. We conclude that acid inhibits TRPV3 function from the extracellular side but facilitates it from the intracellular side. These novel mechanisms of TRPV3 proton sensing can offer new insights into the role of TRPV3 in the regulation of epidermal barrier permeability and skin disorders under conditions of tissue acidosis.     

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1)

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.     

Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.     

A gating mutation in the internal pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.     

Inhibition of KCa2.2 and KCa2.3 channel currents by protonation of outer pore histidine residues

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed K_Ca2.2 (SK2) and K_Ca2.3 (SK3) channel currents, with K_Ca2.3 (pIC₅₀ of ∼6.8) being approximately fourfold more sensitive than K_Ca2.2 (pIC₅₀ of ∼6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. K_Ca2.2 and K_Ca2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with K_Ca2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of K_Ca2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in K_Ca2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

Modulation of TRPV4 gating by intra- and extracellular Ca

We have studied the modulation of gating properties of the Ca²⁺-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca²⁺. Increasing the concentration of extracellular Ca²⁺ ([Ca²⁺]_e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca²⁺]_e. It was also much slower in the presence of [Ca²⁺]_e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca²⁺, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.     

Extracellular protons titrate voltage gating of a ligand-gated ion channel

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.     

Independent Contribution of Extracellular Proton Binding Sites to ASIC1a Activation

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Outer pore architecture of a Ca2+-selective TRP channel

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 A. Mutating Asp(541), a residue involved in high affinity Ca(2+) binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp(541) displayed a cyclic pattern of reactivity to Ag(+) and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K(+) channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K(+) channels and lined by amino acid side chains rather than main chain carbonyls.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.