Ca2+ toxicity due to reverse Na+/Ca2+ exchange contributes to degeneration of neurites of DRG neurons induced by a neuropathy-associated Nav1.7 mutation.

Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na(+) concentration ([Na(+)]) and intracellular [Ca(2+)] following stimulation with high [K(+)] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca(2+)] transients evoked by high [K(+)] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K(+)] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K(+)] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca(2+) or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K(+)] and 2-DG. These results point to [Na(+)] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca(2+) toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation.

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

NaV1.7 gain-of-function mutations as a continuum: A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders.

Gain-of-function mutations of Na(V)1.7 have been shown to produce two distinct disorders: Na(V)1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; Na(V)1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel Na(V)1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4-S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (-7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in Na(V)1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a Na(V)1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

PDGF-stimulated calcium influx changes during in vitro cell transformation.

Platelet Derived Growth Factor (PDGF)-stimulated intracellular calcium signals were obtained from preneoplastic clones derived from C3H 10T1/2 mouse fibroblasts during a four-week transformation assay. By the end of the assay, when transformed colonies of cells were becoming apparent, the PDGF-stimulated calcium signal had reduced significantly. In addition, whole-cell patch-clamp electrophysiology measurements indicated a marked reduction in expression of T-type calcium channels. These observations demonstrate that PDGF-induced calcium metabolism changes as initiated cells progress to the transformed phenotype.

Competence induction by PDGF requires sustained calcium influx by a mechanism distinct from storage-dependent calcium influx.

The significance and mechanism of extracellular calcium influx in the stimulation by PDGF of cell replication was investigated in density-arrested C3H 10T1/2 mouse fibroblasts. PDGF consistently stimulated a biphasic increase in the [Ca2+]i composed of a rapid transient release of calcium from intracellular storage sites followed by a sustained elevation, significantly greater than prestimulated levels, which was dependent upon the [Ca2+]e and persisted for at least 1 h. The percentage of cells incorporating [3H]-TdR into DNA after stimulation with PDGF+insulin was closely correlated with the magnitude of the sustained [Ca2+]i increase and to the [Ca2+]e. Selective inhibition of the sustained [Ca2+]i increase, by blocking calcium influx with La3+, completely inhibited progression to S phase without affecting the release of calcium from intracellular storage sites. Progression to S phase was inhibited by La3+ or the omission of added extracellular calcium only during PDGF exposure and not during treatment with insulin. PDGF-induced calcium influx was completely inhibited by La3+ whereas storage-dependent calcium influx (SDCI) induced by thapsigargin was unaffected. Pretreatment with TPA, forskolin, dibutyryl-cAMP, dibutyryl-cGMP, nifedipine, and TMB-8 had no effect on PDGF-induced calcium influx. These data suggest that the induction of replicative competence by PDGF is dependent upon the maintenance of a sustained increase in the intracellular calcium concentration due to the influx of extracellular calcium through a calcium influx pathway distinct from SDCI.

Expression of voltage-gated calcium channels correlates with PDGF-stimulated calcium influx and depends upon cell density in C3H 10T1/2 mouse fibroblasts.

Platelet derived growth factor (PDGF) mobilizes multiple calcium pools in the C3H 10T1/2 mouse fibroblast, including a sustained influx of extracellular calcium. We have used the whole cell patch-clamp technique to directly test for a role of plasma membrane calcium channels in this influx. Whole-cell patch-clamp recordings revealed a voltage-gated calcium channel with gating properties consistent with a 'T-type' designation. This phenotype of the C3H 10T1/2 fibroblasts was dependent upon the cell density in culture. The fraction of cells expressing calcium channels was low (< 10%) in subconfluent culture but rose to approximately 50% as the cells established a confluent monolayer. The magnitude of the PDGF-stimulated sustained calcium influx component measured using Fura-2 increased in parallel with the expression of calcium current. We interpret these results to support the hypothesis that T-type voltage-gated calcium channels contribute to the PDGF-stimulated intracellular calcium signals in these cells.

Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells.

BACKGROUND: Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. RESULTS: Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. CONCLUSIONS: These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.

Characterization of ion channels seen in subconfluent human dermal fibroblasts.

1. Ion channels expressed in human dermal fibroblasts are characterized using the patch-clamp technique. 2. A number of different ion channels were found but their expression occurred at various frequencies. The most commonly found phenotype was the expression of voltage-gated K+ current. This 'typical' K+ current was seen in about 60% of the cells recorded. 3. Subtypes of voltage-gated K+ channels could be discerned by differences in gating kinetics. One has fast inactivation and resembles the 'A' K+ current. Additional subtypes were sometimes discerned based on activation kinetics. 4. The large-conductance Ca(2+)-activated K+ channel (maxi-K+) could be found in nearly every cell but required large depolarizations to activate using the standard Ca(2+)-buffered pipette solution (10(-8) M [Ca2+]i). 5. Inward rectifier K+ channels were seen in a low percentage of cells. The inward rectifier K+ current was sensitive to 'wash-out' if guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was included in the pipette solution dialysing the cell. 6. Tetrodotoxin (TTX)-sensitive voltage-gated Na+ channels were seen but in a lower number of cells recorded, about 20%. Evidence for subtypes of Na+ channels were sometimes seen based on differences in gating kinetics. 7. An ATP-dependent osmotically activated Cl- current was also found. This current showed some outward rectification but was otherwise voltage independent. 8. In addition, a cell-to-cell contact-associated K+ current was described. This current was linear over the voltage ranges used and whose gating correlated with the existence of gap junctions. 9. These currents were characterized to determine the baseline behaviour of unstimulated cells and to compare to bradykinin-stimulated cells described in the following paper. As unexcitable cells, human dermal fibroblasts are capable of expressing a surprising diversity of ion channel phenotypes and of ion channel modulations.

Acute electrophysiological responses of bradykinin-stimulated human fibroblasts.

1. Acute responses to bradykinin in human dermal fibroblasts were studied at 20-24 degrees C using both the patch-clamp technique to monitor ion currents and Fura-2 fluorescence to monitor [Ca2+]i. 2. During subconfluent culture, human dermal fibroblasts can express a diversity of ion channels as described in the preceding paper. 3. When GTP (1 mM) was included in the pipette solution, two additional ion channel populations were transiently augmented in response to bradykinin stimulation. 4. The first is a component of outwardly rectifying current which reached maximal induction within 10-15 s after bradykinin addition (1 microM) and then decayed back to near baseline over 60 s. 5. Ion substitution experiments combined with tail current analysis indicate that the outward current is carried predominantly by K+. 6. Video imaging of single-cell Fura-2 fluorescence from both intact cells and patch-clamped cells showed temporal correlation of the K+ current modulation and the Ca2+ transients in response to bradykinin stimulation. 7. The calcium ionophore, ionomycin, caused both an increase in intracellular calcium and the augmentation of the outward K+ current. The amount of additional K+ current was correlated with [Ca2+]i levels and could be elicited even without the presence of GTP in the pipette. 8. Apamin, a blocker of Ca(2+)-activated K+ channels, inhibited (at 1 microM) the ionomycin-induced modulation of K+ current. 9. In addition, an inward current was transiently induced in response to bradykinin. This current was strictly dependent on the presence of GTP in the pipette solution. This current showed little voltage dependence, as evidenced by a linear current vs. voltage relation, and a reversal potential near but measurably more positive than 0 mV. 10. This current could be decoupled from the Ca2+ transient and be irreversibly induced by including GTP gamma S (100 microM) in the pipette solution. 11. Ion substitution experiments show that this is a non-specific cation channel. This current prefers monovalents but exhibits a small permeability to divalents. 12. GTP gamma S-induced single channels from isolated outside-out patches showed similar ion selectivity and voltage dependence. These channels are 32 pS in size with an estimated reversal potential of 17 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of voltage-dependent Na+ current in cell-fusion hybrids containing activated c-Ha-ras.

The electrophysiological properties of EJ (human bladder carcinoma), GM2291 (human fetal lung fibroblast), and of three hybrid cell lines obtained from their cell fusion were investigated using the patch-clamp technique. GM2291 cells, which are nontumorigenic, express voltage-dependent Na+ channels. The pharmacology and gating properties of the Na+ channels in GM2291 cells are distinct from neuronal and cardiac Na+ channels. EJ cells, which are tumorigenic and contain activated c-Ha-ras, express inward rectifier K+ channels. The three cell-fusion hybrid lines, named 145 (nontumorigenic), 145L (nontumorigenic but morphologically altered), and 147TR2 (fully tumorigenic segregant), have been previously shown to express levels of activated c-Ha-ras similar to those of the EJ parental line. Voltage-dependent Na+ channels were observed in none of the hybrid cell lines, while inward rectifier K+ channels were observed in each of the hybrid cell lines. The possibility that c-Ha-ras inhibits expression of a voltage-dependent Na+ channel is discussed.

Ca2+ influx via T-type channels modulates PDGF-induced replication of mouse fibroblasts.

The role of low-threshold voltage-gated calcium channels (VGCC) in modulating extracellular calcium influx and proliferation was investigated in platelet-derived growth factor (PDGF)-stimulated C3H/10T1/2 mouse fibroblasts. Previous studies demonstrated that cell cycle progression after PDGF stimulation was dependent on extracellular calcium influx producing a sustained increase in the intracellular calcium concentration. In this study, PDGF-induced calcium influx, the sustained intracellular calcium increase, and progression to S phase were inhibited by nordihydroguariaretic acid (NDGA), an inhibitor of calcium influx through VGCC. With the use of the whole cell patch-clamp technique to measure calcium currents, NDGA inhibited inward calcium current through low-threshold VGCC, the only VGCC expressed in C3H/10T1/2 fibroblasts. The inhibitory effects of NDGA on calcium influx and cell proliferation each had a mean inhibitory dose of 2-3 microM. Although NDGA also effectively inhibits cyclooxygenase and lipoxygenase, the addition of prostaglandins or leukotrienes could not reverse this inhibition nor could it be replicated by other antioxidants. These data support the hypothesis that low-threshold VGCC can mediate extracellular calcium influx on the stimulation of cell proliferation by PDGF.

Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts.

Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several hereditary disorders of bone development, with specific FGFR3 mutations causing achondroplasia (Ach) and thanatophoric dysplasia (TD). However, for none of these mutations has the defect in receptor function been demonstrated directly and, therefore, for none has the pathophysiological mechanism of the disease been defined. Using our established techniques for single-cell ratiometric real-time calcium image analysis, we defined the nature of the basic fibroblast growth factor (bFGF)-induced calcium signal in human diploid fibroblasts, and, in blinded studies, have analyzed the bFGF-induced signals from 18 independent fibroblast cell lines, including multiple lines from patients with known mutant alleles of FGFR3 and syndromes of Ach or TD. Control cells responded with transient increases in intracellular calcium, with many cells showing oscillatory calcium waves. Homozygous Ach cell lines failed to signal, whereas heterozygous Ach lines responded nearly normally. We observed heterogeneous signals in TD heterozygotes: the unresponsive lines all turned out to carry TD1 alleles, whereas all responsive lines had TD2 alleles. Since FGFR1, 2 and 3 receptors are known to be expressed in fibroblasts, our results suggest that specific mutant FGFR3 alleles can function in a dosage-dependent dominant-negative fashion to inactivate FGFR signaling.

Calcium is permeable through a maitotoxin-activated nonselective cation channel in mouse L cells.

The shellfish poison maitotoxin causes the irreversible opening of nonselective cation channels in mouse L cell fibroblasts, consistent with the action of this toxin in other cell types and the previously demonstrated existence of 28-pS voltage-insensitive nonselected cation channels that are activated by platelet-derived growth factor in these cells. Toxin-induced opening of these nonselective cation channels led to increases of intracellular calcium and secondary activation of calcium-activated potassium channel. These effects were completely dependent on influx of extracellular calcium, supporting the conclusion that the maitotoxin-activated nonselective cation channels are permeable to calcium as well as to sodium and potassium. The implication of this finding is that calcium signaling through this channel underlies its links into the growth factor response.

Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.

TrpC1 appears to be a store-operated channel (SOC) when expressed in mammalian cells. In the present study, TrpC1 was expressed in Sf9 insect cells using the baculovirus expression system. Expression of TrpC1 caused an increase in basal cytosolic free Ca2+ concentration ([Ca2+]i) as a function of post-infection time. Basal Ba2+ influx, an index of plasmalemmal Ca2+ permeability, was also increased and was blocked by La3+. Although the thapsigargin-induced change in [Ca2+]i was greater in TrpC1-expressing cells than controls, Ba2+ influx was unaffected by thapsigargin. Whole-cell membrane currents recorded in TrpC1-expressing cells increased as a function of post-infection time and were (1) inwardly rectifying in symmetrical sodium gluconate solutions, (2) non-selective with respect to Na+, Ca2+ and Ba2+, and (3) blocked by La3+. Furthermore TrpC1 currents were unaffected by (1) thapsigargin, (2) dialysis of the cell with Ins(1,4,5)P3 or (3) dialysis of the cell with solutions containing high concentrations of the Ca2+ chelator, EGTA. These results suggest that TrpC1 forms non-selective cation channels that are constitutively active when expressed in Sf9 cells, but insensitive to depletion of the internal Ca2+ stores. Thus TrpC1 may be a subunit of a SOC which alone can form functional channels in Sf9 cells, but which requires additional subunits or cytoplasmic factors present in mammalian cells for expression of SOC activity.

Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts.

Maitotoxin (MTX), a potent cytolytic agent, activates Ca(2+) entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca(2+) concentration that depended on extracellular Ca(2+) and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-D-glucamine (167 Da). Thus MTX initially activates Ca(2+)-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X(7) receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore.

Stimulation of Drosophila TrpL by capacitative Ca2+ entry.

Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+>Sr2+>> Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE.

Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

Regulation of Drosophila TRPL channels by immunophilin FKBP59.

Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel.