Pregabalin Silences Oxaliplatin-Activated Sensory Neurons to Relieve Cold Allodynia.

Oxaliplatin is a platinum-based chemotherapeutic agent that causes cold and mechanical allodynia in up to 90% of patients. Silent Nav1.8-positive nociceptive cold sensors have been shown to be unmasked by oxaliplatin, and this event has been causally linked to the development of cold allodynia. We examined the effects of pregabalin on oxaliplatin-evoked unmasking of cold sensitive neurons using mice expressing GCaMP-3 in all sensory neurons. Intravenous injection of pregabalin significantly ameliorates cold allodynia, while decreasing the number of cold sensitive neurons by altering their excitability and temperature thresholds. The silenced neurons are predominantly medium/large mechano-cold sensitive neurons, corresponding to the "silent" cold sensors activated during neuropathy. Deletion of α2δ1 subunits abolished the effects of pregabalin on both cold allodynia and the silencing of sensory neurons. Thus, these results define a novel, peripheral inhibitory effect of pregabalin on the excitability of "silent" cold-sensing neurons in a model of oxaliplatin-dependent cold allodynia.

Calcium imaging for analgesic drug discovery.

Somatosensation and pain are complex phenomena involving a rangeofspecialised cell types forming different circuits within the peripheral and central nervous systems. In recent decades, advances in the investigation of these networks, as well as their function in sensation, resulted from the constant evolution of electrophysiology and imaging techniques to allow the observation of cellular activity at the population level both in vitro and in vivo. Genetically encoded indicators of neuronal activity, combined with recent advances in DNA engineering and modern microscopy, offer powerful tools to dissect and visualise the activity of specific neuronal subpopulations with high spatial and temporal resolution. In recent years various groups developed in vivo imaging techniques to image calcium transients in the dorsal root ganglia, the spinal cord and the brain of anesthetised and awake, behaving animals to address fundamental questions in both the physiology and pathophysiology of somatosensation and pain. This approach, besides giving unprecedented details on the circuitry of innocuous and painful sensation, can be a very powerful tool for pharmacological research, from the characterisation of new potential drugs to the discovery of new, druggable targets within specific neuronal subpopulations. Here we summarise recent developments in calcium imaging for pain research, discuss technical challenges and advances, and examine the potential positive impact of this technique in early preclinical phases of the analgesic drug discovery process.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies.

Ion channels play crucial roles in physiology by modulation of cellular functions that include electrical excitability, secretion, cell migration, and gene transcription. They are an important target class for drug discovery and have historically been targeted using small molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative forms of biologics. Antibodies display high specificity, selectivity, and affinity for their target antigen, thus having the potential to target ion channels very precisely. Nonetheless, isolating antibodies to ion channels is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery and due to the complexities of screening for function. In this overview, we focus on an array of screening methods, ranging from direct antibody binding screens to complex electrophysiological assays, and describe how these assays can be used to identify functional monoclonal antibodies. We also provide some insights into specific considerations which are required to enable these screens to be used for antibody drug discovery. © 2018 by John Wiley & Sons, Inc.

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice.

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel ß3 (Scn3b), rather than the ß1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

Recombinant expression and in vitro characterisation of active Huwentoxin-IV.

Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV(G36)(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.

Bradykinin controls pool size of sensory neurons expressing functional δ-opioid receptors.

Analgesics targeting the δ-opioid receptor (DOR) may lead to fewer side effects than conventional opioid drugs, which mainly act on µ-opioid receptors (MOR), because of the less abundant expression of DOR in the CNS compared with MOR. Analgesic potential of DOR agonists increases after inflammation, an effect that may be mediated by DOR expressed in the peripheral sensory fibers. However, the expression of functional DOR at the plasma membrane of sensory neurons is controversial. Here we have used patch-clamp recordings and total internal reflection fluorescence microscopy to study the functional expression of DOR in sensory neurons from rat trigeminal (TG) and dorsal root ganglia (DRG). Real-time total internal reflection fluorescence microscopy revealed that treatment of TG and DRG cultures with the inflammatory mediator bradykinin (BK) caused robust trafficking of heterologously expressed GFP-tagged DOR to the plasma membrane. By contrast, treatment of neurons with the DOR agonist [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) caused a decrease in the membrane abundance of DOR, suggesting internalization of the receptor after agonist binding. Patch-clamp experiments revealed that DADLE inhibited voltage-gated Ca(2+) channels (VGCCs) in 23% of small-diameter TG neurons. Pretreatment with BK resulted in more than twice as many DADLE responsive neurons (54%) but did not affect the efficacy of VGCC inhibition by DADLE. Our data suggest that inflammatory mediator-induced membrane insertion of DOR into the plasma membrane of peripheral sensory neurons may underlie increased DOR analgesia in inflamed tissue. Furthermore, the majority of BK-responsive TG neurons may have a potential to become responsive to DOR ligands in inflammatory conditions.

Perforated whole-cell patch-clamp recording.

Perforated whole-cell patch-clamp is a variant of the patch-clamp technique used to measure the sum activity of ion channels in the plasma membrane of a single cell. Its defining feature is that electrical access to the cell is obtained through inclusion of a pore-forming antibiotic in the patch pipette which perforates the sealed patch of membrane in contact with the patch pipette. The antibiotic pores allow equilibration of small monovalent ions between the patch pipette and the cytosol whilst maintaining endogenous levels of divalent ions such as Ca(2+) and signalling molecules such as cAMP. Therefore, the perforated patch-clamp technique is ideal for studying ion channels whilst maintaining the integrity of second messenger signalling cascades. Other benefits of using perforated patch-clamp over conventional patch-clamp include reduced current rundown and stable whole-cell recording lasting >1 h. In this chapter, the application of the perforated patch-clamp technique for the study of heterologously expressed Kv7 potassium channels will be discussed in detail including benefits and limitations of the technique.

Potency optimization of Huwentoxin-IV on hNav1.7: a neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena.

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNav1.7 (IC50 determined herein as 17 ± 2 nM). Nav1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure-function study into Nav1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu(1), Glu(4), Phe(6) and Tyr(33) were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNav1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNav1.5. Peptide 47 (Gly(1), Gly(4), Trp(33)-HwTx) demonstrated the largest potency increase on hNav1.7 (IC50 0.4 ± 0.1 nM).

Novel mutations mapping to the fourth sodium channel domain of Nav1.7 result in variable clinical manifestations of primary erythromelalgia.

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype-phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.

Neurological perspectives on voltage-gated sodium channels.

The activity of voltage-gated sodium channels has long been linked to disorders of neuronal excitability such as epilepsy and chronic pain. Recent genetic studies have now expanded the role of sodium channels in health and disease, to include autism, migraine, multiple sclerosis, cancer as well as muscle and immune system disorders. Transgenic mouse models have proved useful in understanding the physiological role of individual sodium channels, and there has been significant progress in the development of subtype selective inhibitors of sodium channels. This review will outline the functions and roles of specific sodium channels in electrical signalling and disease, focusing on neurological aspects. We also discuss recent advances in the development of selective sodium channel inhibitors.

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells.

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons.

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and G(i/o) proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca(2+) stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca(2+) stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.

Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis.

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na(+) and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na(+) absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na(+) absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface-crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K(+) conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na(+) transport, thereby impairing Na(+) absorption (and as a consequence, Cl(-) and water absorption) across the inflamed mucosa.

M channel enhancers and physiological M channel block.

M-type (Kv7, KCNQ) K(+) channels control the resting membrane potential of many neurons, including peripheral nociceptive sensory neurons. Several M channel enhancers were suggested as prospective analgesics, and targeting M channels specifically in peripheral nociceptors is a plausible strategy for peripheral analgesia. However, receptor-induced inhibition of M channels in nociceptors is often observed in inflammation and may contribute to inflammatory pain. Such inhibition is predominantly mediated by phospholipase C. We investigated four M channel enhancers (retigabine, flupirtine, zinc pyrithione and H(2)O(2)) for their ability to overcome M channel inhibition via two phospholipase C-mediated mechanisms, namely depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a rise in intracellular Ca(2+) (an action mediated by calmodulin). Data from overexpressed Kv7.2/Kv7.3 heteromers and native M currents in dorsal root ganglion neurons suggest the following conclusions. (i) All enhancers had a dual effect on M channel activity, a negative shift in voltage dependence and an increase of the maximal current at saturating voltages. The enhancers differed in their efficacy to produce these effects. (ii) Both PIP(2) depletion and Ca(2+)/calmodulin strongly reduced the M current amplitude; however, at voltages near the threshold for M channel activation (-60 mV) all enhancers were able to restore M channel activity to a control level or above, while at saturating voltages the effects were more variable. (iii) Receptor-mediated inhibition of M current in nociceptive dorsal root ganglion neurons did not reduce the efficacy of retigabine or flupirtine to hyperpolarize the resting membrane potential. In conclusion, we show that all four M channel enhancers tested could overcome both PIP(2) and Ca(2+)-calmodulin-induced inhibition of Kv7.2/7.3 at voltages close to the threshold for action potential firing (-60 mV) but generally had reduced efficacy at a saturating voltage (0 mV). We suggest that the efficacy of an M channel enhancer to shift the voltage dependence of activation may be most important for rescuing M channel function in sensory neurons innervating inflamed tissue.

Transcriptional control of KCNQ channel genes and the regulation of neuronal excitability.

Regulation of the resting membrane potential and the repolarization of neurons are important in regulating neuronal excitability. The potassium channel subunits Kv7.2 and Kv7.3 play a key role in stabilizing neuronal activity. Mutations in KCNQ2 and KCNQ3, the genes encoding Kv7.2 and Kv7.3, cause a neonatal form of epilepsy, and activators of these channels have been identified as novel antiepileptics and analgesics. Despite the observations that regulation of these subunits has profound effects on neuronal function, almost nothing is known about the mechanisms responsible for controlling appropriate expression levels. Here we identify two mechanisms responsible for regulating KCNQ2 and KCNQ3 mRNA levels. We show that the transcription factor Sp1 activates expression of both KCNQ2 and KCNQ3, whereas the transcriptional repressor REST (repressor element 1-silencing transcription factor) represses expression of both of these genes. Furthermore, we show that transcriptional regulation of KCNQ genes is mirrored by the correlated changes in M-current density and excitability of native sensory neurons. We propose that these mechanisms are important in the control of excitability of neurons and may have implications in seizure activity and pain.

The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl- channels.

Bradykinin (BK) is an inflammatory mediator and one of the most potent endogenous pain-inducing substances. When released at sites of tissue damage or inflammation, or applied exogenously, BK produces acute spontaneous pain and causes hyperalgesia (increased sensitivity to potentially painful stimuli). The mechanisms underlying spontaneous pain induced by BK are poorly understood. Here we report that in small nociceptive neurons from rat dorsal root ganglia, BK, acting through its B2 receptors, PLC, and release of calcium from intracellular stores, robustly inhibits M-type K+ channels and opens Ca2+-activated Cl- channels (CaCCs) encoded by Tmem16a (also known as Ano1). Summation of these two effects accounted for the depolarization and increase in AP firing induced by BK in DRG neurons. Local injection of inhibitors of CaCC and specific M-channel openers both strongly attenuated the nociceptive effect of local injections of BK in rats. These results provide a framework for understanding spontaneous inflammatory pain and may suggest new drug targets for treatment of such pain.

Understanding inflammatory pain: ion channels contributing to acute and chronic nociception.

Inflammatory pain results from the increased excitability of peripheral nociceptive sensory fibres produced by the action of inflammatory mediators. This excitatory effect, in turn, is a result of the altered activity of ion channels within affected sensory fibres. This review will consider the molecular consequences of inflammation within the peripheral nerves with particular focus on the effects of different inflammatory mediators on the ion channels in sensory neurons. We will discuss the main signalling pathways triggered in neurons by inflammatory mediators; the ionic mechanisms underlying inflammatory hyperalgesia and spontaneous inflammatory pain and finally will briefly consider ion channels underlying pain in chronic inflammation.

A voltage-dependent Ca2+ influx pathway regulates the Ca2+-dependent Cl(-) conductance of renal IMCD-3 cells.

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca2+-dependent Cl(-) current (I(CLCA)). Here we report that I(CLCA) activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al3+-sensitive, voltage-dependent, Ca2+ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (-60 mV), ICLCA was found to be inactive and resting currents were predominantly K+ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of ICLCA (T(0.5) approximately 500 s), while repolarization in turn resulted in a monoexponential decay in I(CLCA) (T (0.5) approximately 100 s). The activation of I(CLCA) by depolarization was reduced by lowering extracellular Ca2+ and completely inhibited by buffering cytosolic Ca2+ with EGTA, suggesting a role for Ca2+ influx in the activation of I(CLCA). However, raising bulk cytosolic Ca2+ at -60 mV did not produce sustained I(CLCA) activity. Therefore I(CLCA) is dependent on both an increase in intracellular Ca2+ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca2+ influx pathway that displays equal permeability to Ca2+ and Ba2+ ions and that is blocked by extracellular Al3+ and La3+. Furthermore, Al3+ completely and reversibly inhibited depolarization-induced activation of ICLCA, thereby directly linking Ca2+ influx to activation of I(CLCA). We speculate that during sustained membrane depolarization, calcium influx activates ICLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.