Christopher J. Benson

The mammalian sodium channel BNC1 is required for normal touch sensation

Of the vertebrate senses, touch is the least understood at the molecular level. The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.

Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons

Acidic extracellular solution activates transient H+-gated currents in dorsal root ganglion (DRG) neurons. The biophysical properties of three degenerin/epithelial sodium (DEG/ENaC) channel subunits (BNC1, ASIC, and DRASIC), and their expression in DRG, suggest that they might underlie these H+-gated currents and function as sensory transducers. However, it is uncertain which of these DEG/ENaC subunits generate the currents, and whether they function as homomultimers or heteromultimers. We found that the biophysical properties of transient H+-gated currents from medium to large mouse DRG neurons differed from BNC1, ASIC, or DRASIC expressed individually, but were reproduced by coexpression of the subunits together. To test the contribution of each subunit, we studied DRG from three strains of mice, each bearing a targeted disruption of BNC1, ASIC, or DRASIC. Deletion of any one subunit did not abolish H+-gated currents, but altered currents in a manner consistent with heteromultimerization of the two remaining subunits. These data indicate that combinations of two or more DEG/ENaC subunits coassemble as heteromultimers to generate transient H+-gated currents in mouse DRG neurons.

Acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons

Cardiac afferents are sensory neurons that mediate angina, pain that occurs when the heart receives insufficient blood supply for its metabolic demand (ischemia). These neurons display enormous acid-evoked depolarizing currents, and they fire action potentials in response to extracellular acidification that accompanies myocardial ischemia. Here we show that acid-sensing ion channel 3 (ASIC3), but no other known acid-sensing ion channel, reproduces the functional features of the channel that underlies the large acid-evoked current in cardiac afferents. ASIC3 and the native channel are both especially sensitive to pH, interact similarly with Ca(2+), and gate rapidly between closed, open, and desensitized states. Particularly important is the ability of ASIC3 and the native channel to open at pH 7, a value reached in the first few minutes of a heart attack. The steep activation curve suggests that the channel opens when four protons bind. We propose that ASIC3, a member of the degenerin channel (of Caenorhabditis elegans)/epithelial sodium channel family of ion channels, is the sensor of myocardial acidity that triggers cardiac pain, and that it might be a useful pharmaceutical target for treating angina.

Acid-Evoked Currents in Cardiac Sensory Neurons A Possible Mediator of Myocardial Ischemic Sensation

Sensory neurons that innervate the heart sense ischemia and mediate angina. To use patch-clamp methods to study ion channels on these cells, we fluorescently labeled cardiac sensory neurons (CSNs) in rats so that they could later be identified in dissociated primary culture of either nodose or dorsal root ganglia (DRG). Currents evoked by a variety of different agonists imply the importance of lowered pH (</=7.0) in signaling ischemia. Acidic pH evoked extremely large depolarizing current in almost all cardiac afferent neurons from the DRG (CDRGNs). In contrast, only about half of the unlabeled DRG neurons responded to acid, and their current amplitudes were much less than that in CDRGNs. In all respects tested--kinetics, selectivity, and pharmacology--the acid-evoked current was similar to that of previously described native and cloned acid-sensing ion channels. Cardiac afferents from the nodose ganglia differed from CDRGNs in having smaller acid-evoked currents but clearly larger currents evoked by ATP. Serotonin, acetylcholine, bradykinin, and adenosine elicited currents in fewer CSNs than did ATP or lowered pH, and the currents were relatively small. Capsaicin, an activator of small nociceptive sensory neurons that innervate skin, evoked only small and rare currents in CDRGNs. The extremely large amplitude and prevalent expression of acid-evoked current in CSNs imply a critical role for acidity in sensation associated with myocardial ischemia.

Neuropeptide FF and FMRFamide Potentiate Acid-Evoked Currents from Sensory Neurons and Proton-Gated DEG/ENaC Channels

Acidosis is associated with inflammation and ischemia and activates cation channels in sensory neurons. Inflammation also induces expression of FMRFamidelike neuropeptides, which modulate pain. We found that neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe amide) and FMRFamide (Phe-Met-Arg-Phe amide) generated no current on their own but potentiated H+-gated currents from cultured sensory neurons and heterologously expressed ASIC and DRASIC channels. The neuropeptides slowed inactivation and induced sustained currents during acidification. The effects were specific; different channels showed distinct responses to the various peptides. These results suggest that acid-sensing ion channels may integrate multiple extracellular signals to modify sensory perception.

ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation

Abstract Peripheral initiators of muscle pain are virtually unknown, but likely key to development of chronic pain after muscle insult. The current study tested the hypothesis that ASIC3 in muscle is necessary for development of cutaneous mechanical, but not heat, hyperalgesia induced by muscle inflammation. Using mechanical and heat stimuli, we assessed behavioral responses in ASIC3−/− and ASIC3+/+ mice after induction of carrageenan muscle inflammation. ASIC3−/− mice did not develop cutaneous mechanical hyperalgesia after muscle inflammation when compared to ASIC3+/+ mice; heat hyperalgesia developed similarly between groups. We then tested if the phenotype could be rescued in ASIC3−/− mice by using a recombinant herpes virus vector to express ASIC3 in skin (where testing occurred) or muscle (where inflammation occurred). Infection of mouse DRG neurons with ASIC3‐encoding virus resulted in functional expression of ASICs. Injection of ASIC3‐encoding virus into muscle or skin of ASIC3−/− mice resulted in ASIC3 mRNA in DRG and protein expression in DRG and the peripheral injection site. Injection of ASIC3‐encoding virus into muscle, but not skin, resulted in development of mechanical hyperalgesia similar to that observed in ASIC3+/+ mice. Thus, ASIC3 in primary afferent fibers innervating muscle is critical to development of hyperalgesia that results from muscle insult.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

The Ion Channel ASIC2 Is Required for Baroreceptor and Autonomic Control of the Circulation

Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of circulation. Our goal was to test the hypothesis that a member of the acid-sensing ion channel (ASIC) subfamily of the DEG/ENaC superfamily is an important determinant of the arterial baroreceptor reflex. We found that aortic baroreceptor neurons in the nodose ganglia and their terminals express ASIC2. Conscious ASIC2 null mice developed hypertension, had exaggerated sympathetic and depressed parasympathetic control of the circulation, and a decreased gain of the baroreflex, all indicative of an impaired baroreceptor reflex. Multiple measures of baroreceptor activity each suggest that mechanosensitivity is diminished in ASIC2 null mice. The results define ASIC2 as an important determinant of autonomic circulatory control and of baroreceptor sensitivity. The genetic disruption of ASIC2 recapitulates the pathological dysautonomia seen in heart failure and hypertension and defines a molecular defect that may be relevant to its development.

ASIC2 Subunits Target Acid-Sensing Ion Channels to the Synapse via an Association with PSD-95

Acid-sensing ion channel-1a (ASIC1a) mediates H+-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not coimmunoprecipitate with PSD-95. We found that ASIC2 and ASIC1a associated in brain, and through its interaction with PSD-95, ASIC2 increased ASIC1a localization in dendritic spines. Consistent with earlier work showing that acidic pH elevated spine [Ca2+]i by activating ASIC1a, loss of ASIC2 decreased the percentage of spines responding to acid. Moreover, like a reduction of ASIC1a, the number of spine synapses fell in ASIC2−/− neurons. These results indicate that ASIC2 facilitates ASIC1a localization and function in dendritic spines and suggest that the two subunits work in concert to regulate neuronal function.

Acid-sensing ion channels contribute to transduction of extracellular acidosis in rat carotid body glomus cells

Carotid body chemoreceptors sense hypoxemia, hypercapnia, and acidosis and play an important role in cardiorespiratory regulation. The molecular mechanism of pH sensing by chemoreceptors is not clear, although it has been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K+ current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using the whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward current in glomus cells that was inhibited by the acid-sensing ion channel (ASIC) blocker amiloride, absent in Na+-free bathing solution, and enhanced by either Ca2+-free buffer or addition of lactate. In addition, ASIC1 and ASIC3 were shown to be expressed in rat carotid body by quantitative PCR and immunohistochemistry. In the current-clamp mode, extracellular acidosis evoked both a transient and sustained depolarizations. The initial transient component of depolarization was blocked by amiloride, whereas the sustained component was eliminated by removal of K+ from the pipette solution and partially blocked by the TASK (tandem-p-domain, acid-sensitive K+ channel) blockers anandamide and quinidine. The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels.