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Polycystic kidney disease (PKD), a disease characterized by the enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. Transient receptor potential Vanilloid 4 (TRPV4), a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations. Graphical Abstract Abstract graph showing a schematic representation of a wild-type TRPV4 channel compared to the increased activity of a mutant TRPV4 channel carrying one of the two TRPV4 mutations located in the amino-terminal domain and found in a patient cohort diagnosed with polycystic kidney disease.Abstract graph showing a schematic representation of a wild-type TRPV4 channel compared to the increased activity of a mutant TRPV4 channel carrying one of the two TRPV4 mutations located in the amino-terminal domain and found in a patient cohort diagnosed with polycystic kidney disease. [ABSTRACT FROM AUTHOR]

The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 [Angstrom] for the inner pore of TRPV1 channels.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for [Ca.sup.2+]. Not surprisingly, they are blocked by a number of [Ca.sup.2+] channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance [Ca.sup.2+]-activated [K.sup.+] channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the $4-$5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway. KEY WORDS: CNG channels * dequalinium * voltage dependence * block * gating

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher; M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of [Na.sup.2+], but their high [Ca.sup.2+] permeability is also important for mediating the response to pain. In particular, [Ca.sup.2+] influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Nueroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, [Ca.sup.2+]/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by [Mg.sup.2+], reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by [Ca.sup.2+] alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to [Ca.sup.2+]. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a [Ca.sup.2+]-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel N[H.sub.2]terminal domain demonstrated that a stretch of ~30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a [Ca.sup.2+]-dependent manner. The Physiological response to pain involves an influx of [Ca.sup.2+] through TRPV1. Our results indicate that this [Ca.sup.2+] influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin. KEY WORDS: desensitization * capsaicin * TRPV1 * calcium * calmodulin

Cyclic nucleotide--gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance [Ca.sup.2+]-activated [K.sup.+] channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a [K.sub.1/2] [approximately equal to] 190 nM and CNGA1+CNGB1 channels with a [K.sub.1/2] [approximately equal to] 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a z[delta] [approximately equal to] 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels. KEY WORDS: dequalinium * block * CNG channels

Differentiation and function of pancreatic [Beta] cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic [Beta] cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in [Beta] cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic [Beta]-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

TRPV4 is a nonselective cationic channel responsive to several physical and chemical stimuli. Defects in TRPV4 channel function result in human diseases, such as skeletal dysplasias, arthropathies, and peripheral neuropathies. Nonetheless, little is known about the role of TRPV4 in other cellular functions, such as nuclear Ca2+ homeostasis or Ca2+‐regulated transcription. Here, we confirmed the presence of the full‐length TRPV4 channel in the nuclei of nonpolarized Madin‐Darby canine kidney cells. Confocal Ca2+ imaging showed that activation of the channel increases cytoplasmic and nuclear Ca2+ leading to translocation of TRPV4 out of the nucleus together with β‐catenin, a transcriptional regulator in the Wnt signaling pathway fundamental in embryogenesis, organogenesis, and cellular homeostasis. TRPV4 inhibits β‐catenin transcriptional activity through a direct interaction dependent upon channel activity. This interaction also occurs in undifferentiated osteoblastoma and neuroblastoma cell models. Our results suggest a mechanism in which TRPV4 may regulate differentiation in several cellular contexts. [ABSTRACT FROM AUTHOR]

Abstract Background The capsaicin (vanilloid) receptor, VR1, is an agonist-activated ion channel expressed by sensory neurons that serves as a detector of chemical and thermal noxious stimuli. Results In the present study we investigated the properties of VR1 ion channels expressed in Xenopus oocytes. A VR1 subunit with a FLAG epitope tag at the C-terminus was constructed. When examined for size on an SDS gel, VR1-expressing oocytes produced a doublet corresponding to the size of the monomer and a band at about twice the molecular weight of the monomer. A consensus site for N-linked glycosylation was identified in the primary sequence at position 604. In channels in which the putative glycosylation site was mutated from asparagine to serine (N604S), the larger of the two monomer bands could no longer be detected on the gel. Electrophysiological experiments showed these unglycosylated channels to be functional. The high molecular weight band observed on the gel could represent either a dimer or a monomer conjugated to an unknown factor. To distinguish between these possibilities, we coexpressed a truncated VR1 subunit with full-length VR1. A band of intermediate molecular weight (composed of one full-length and one truncated subunit) was observed. This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors. Conclusions The persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits. This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.