[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM), a Ca2+ sensing protein, is constitutively bound to IQ domains of the C-termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca2+-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of
the hKv7.4 channel in a Ca2+-independent manner but only the long isoform (hKv7.4a) is regulated by Ca2+/CaM. Ca2+/CaM mediate reduction of the hKv7.4a channel by decreasing channel open probability and altering activation kinetics. We
took advantage of a known missense mutation (G321S) which has been linked to progressive hearing loss to further examine the
inhibitory effects of Ca2+/CaM on Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that G321S mutation may destabilize CaM binding
leading to a decrease in the inhibitory effects of Ca2+ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4
channels. The report provides mechanistic insights into critical roles of Ca2+/CaM regulation of Kv7.4 channel in physiological and pathological conditions.
Preview · Article · Oct 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.
Full-text · Article · Feb 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca(2+)-activated K(+) channels (KCa2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca(2+)-dependent. Further, the Ca(2+) dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.
Full-text · Article · Jun 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Spiral ganglion neurons (SGNs) of the eighth nerve serve as the bridge between hair cells and the cochlear nucleus. Hair cells use Cav1.3 as the primary channel for Ca(2+) inflow to mediate transmitter release. In contrast, SGNs are equipped with multiple Ca(2+) channels to mediate Ca(2+)-dependent functions. We examined directly the role of Cav1.3 channels in SGNs using Cav1.3-deficient mice (Cav1.3(-/-)). We revealed a surprising finding that SGNs functionally express the cardiac-specific Cav1.2, as well as neuronal Cav1.3 channels. We show that evoked action potentials recorded from SGNs show a significant decrease in the frequency of firing in Cav1.3(-/-) mice compared with wild-type (Cav1.3(+/+)) littermates. Although Cav1.3 is the designated L-type channel in neurons, whole-cell currents recorded in isolated SGNs from Cav1.3(-/-) mice showed a surprising remnant current with sensitivity toward the dihydropyridine (DHP) agonist and antagonist, and a depolarization shift in the voltage-dependent activation compared with that in the Cav1.3(+/+) mice. Indeed, direct measurement of the elementary properties of Ca(2+) channels, in Cav1.3(+/+) neurons, confirmed the existence of two DHP-sensitive single-channel currents, with distinct open probabilities and conductances. We demonstrate that the DHP-sensitive current in Cav1.3(-/-) mice is derived from Cav1.2 channel activity, providing for the first time, to our knowledge, functional data for the expression of Cav1.2 currents in neurons. Finally, using shRNA gene knockdown methodology, and histological analyses of SGNs from Cav1.2(+/-) and Cav1.3(+/-) mice, we were able to establish the differential roles of Cav1.2 and Cav1.3 in SGNs.
Full-text · Article · May 2014 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of
Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall
functional relevance of the single transmembrane domain peptide on the pore-forming K+ channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with
Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the
properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations
in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched
wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3−/− mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing
frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K+ currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that
KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes
that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable
from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.
[Show abstract][Hide abstract] ABSTRACT: The extracellular potential of excitable and nonexcitable cells with respect to ground is ∼0 mV. One of the known exceptions in mammals is the cochlear duct, where the potential is ∼80-100 mV, called the endocochlear potential (EP). The EP serves as the "battery" for transduction of sound, contributing toward the sensitivity of the auditory system. The stria vascularis (StV) of the cochlear duct is the station where the EP is generated, but the cell-specific roles in the StV are ill defined. Using the intermediate cell (IC)-specific tyrosinase promoter, under the control of diphtheria toxin (DT), we eliminated and/or halted differentiation of neural crest melanocytes after migration to the StV. The ensuing adult transgenic mice are profoundly deaf. Additionally, the EP was abolished. Expression of melanocyte early marker and Kir4.1 in ICs precedes the onset of pigment synthesis. Activation of DT leads to loss of ICs. Finally, in accord with the distinct embryology of retinal pigmented cells, transgenic mice with toxigenic ablation of neural crest-derived melanocytes have intact visual responses. We assert that the tyrosinase promoter is the distinct target for genetic manipulation of IC-specific genes.
Preview · Article · Sep 2013 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Developmental plasticity in spiral ganglion neurons (SGNs) ensues from profound alterations in the functional properties of the developing hair cell (HC). For example, pre-hearing HCs are spontaneously active. However, at the post-hearing stage, HC membrane properties transition to graded receptor potentials. The dendrotoxin (DTX)-sensitive Kv1 channel subunits (Kv1.1, 1.2, 1.6) shape the firing properties and membrane potential of SGNs, and the expression of the channel undergoes developmental changes. Because of the stochastic nature of Kv subunit heteromultimerization, it has been difficult to determine physiologically relevant subunit-specific interactions and their functions in the underlying mechanisms of Kv1 channel plasticity in SGNs. Using Kcna2 null mutant mice, we demonstrate a surprising paradox in changes in the membrane properties of SGNs. The resting membrane potential of Kcna2(-/-) SGNs was significantly hyperpolarized compared to age-matched wild type (WT) SGNs. Analyses of outward currents in the mutant SGNs suggest an apparent ~2-fold increase in outward K(+) currents. We show that in vivo and in vitro heteromultimerization of Kv1.2 and 1.4 α-subunits underlies the striking and unexpected alterations in the properties of SGNs. The results suggest that heteromeric interactions of Kv1.2 and 1.4 dominate the defining features of Kv1 channels in SGNs.
No preview · Article · Jul 2013 · Journal of Neurophysiology
[Show abstract][Hide abstract] ABSTRACT: Rationale:
Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules.
We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes.
Methods and results:
We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling-mediated enhancement of the L-type Ca(2+) current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling.
Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types.
No preview · Article · Apr 2013 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Mechanisms that guide directional migration of neuroblasts from the subventricular zone (SVZ) are not well understood. We report here that endogenous electric currents serve as a guidance cue for neuroblast migration. We identify the existence of naturally occurring electric currents (1.5±0.6 μA/cm(2), average field strength of ∼3 mV/mm) along the rostral migration path in adult mouse brain. Electric fields of similar strength direct migration of neuroblasts from the SVZ in culture and in brain slices. The purinergic receptor P2Y1 mediates this migration. The results indicate that naturally occurring electric currents serve as a new guidance mechanism for rostral neuronal migration.
[Show abstract][Hide abstract] ABSTRACT: Whereas prehearing spiral ganglion neurons (SGNs) rely faithfully on outputs from spontaneously active developing hair cells, the electrical phenotypes of posthearing neurons are shaped by distinct rapid and graded receptor potentials from hair cells. To date, technical difficulties in isolation of fragile posthearing neurons from the rigid bony labyrinth of the inner ear have hindered analyses of the electrical phenotype of SGNs. Therefore, we have recently developed new strategies to isolate posthearing mouse SGNs for functional analyses. Here, we describe the coarse and fine properties of Ca(2+) currents, which sculpt the firing properties of posthearing SGNs. Murine SGNs express multiple Ca(2+) channel currents to enable diverse functions. We have demonstrated that suppression of Ca(2+) currents results in significant hyperpolarization of the resting membrane potential (rmp) of basal SGNs, suggesting that Ca(2+) influx primes rmp for excitation. In contrast, removal of external Ca(2+) has modest effects on rmp of apical SGNs. The blockade of Ca(2+) currents with a mixture of specific blockers attenuates spontaneously active SGNs. Paradoxically, different subtypes of Ca(2+) currents, such as R-type currents, may activate resting outward conductances since blockage of the current results in depolarization of rmp. In keeping with whole-cell current data, single-channel records revealed multiple diverse Ca(2+) channels in SGNs. Additionally, there were differential expressions of distinct Ca(2+) current densities in the apicobasal contour of the adult cochlea. This report provides invaluable insights into Ca(2+)-dependent processes in adult SGNs.
Full-text · Article · Nov 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The objective of the present study is to elucidate the pathogenic role of eicosanoids in myocardial infarction (MI). The accumulation of eicosanoid metabolites in ischaemic myocardium has been demonstrated in animal models and patients with MI, and it occurs in parallel with the development of irreversible cardiac damage. However, the key question that remains unanswered is whether cardiac-generated eicosanoids are the cause or the consequence of cardiac cell damage in MI.
We used a clinically relevant animal model of MI and metabolic profiling to monitor the eicosanoid profile in ischaemic myocardium. We demonstrate that ischaemia induces the generation of prostanoids mainly through the cyclooxygenase (COX)-1 pathway in the myocardium. Cardiac-generated prostanoids, particularly prostaglandin D(2) (PGD(2)), can directly induce apoptosis in cardiac myocytes. This effect involves the up-regulation of the pro-apoptotic gene, Fas ligand (FasL), in a D-type prostanoid receptor-independent manner. The treatment of the MI mice with low-dose aspirin effectively inhibits the ischaemia-induced prostanoid generation and FasL expression in the myocardium, leading to the reduction in cardiac apoptosis following cardiac ischaemia.
Cardiac ischaemia results in COX-1-mediated generation of prostanoids, which by inducing cardiac myocyte apoptosis, contribute to the cardiac cell loss following MI. The benefits of low-dose aspirin treatment in MI may be attributable, in part, to the inhibition of cardiac prostanoid generation and attenuation of apoptosis. Further understanding of the mechanisms underlying prostanoid-induced cardiac apoptosis may be of significant value in designing new therapeutic strategies to prevent aberrant cell loss following MI and subsequent progression to heart failure.
Full-text · Article · Jun 2012 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Evolution has transformed a simple ear with few vestibular maculae into a complex three-dimensional structure consisting of nine distinct endorgans. It is debatable whether the sensory epithelia underwent progressive segregation or emerged from distinct sensory patches. To address these uncertainties we examined the morphological and functional phenotype of trans-differentiated rat hair cells to reveal their primitive or endorgan-specific origins. Additionally, it is uncertain how Atoh1-mediated trans-differentiated hair cells trigger the processes that establish their neural ranking from the vestibulocochlear ganglia. We have demonstrated that the morphology and functional expression of ionic currents in trans-differentiated hair cells resemble those of "ancestral" hair cells, even at the lesser epithelia ridge aspects of the cochlea. The structures of stereociliary bundles of trans-differentiated hair cells were in keeping with cells in the vestibule. Functionally, the transient expression of Na⁺ and I(h) currents initiates and promotes evoked spikes. Additionally, Ca²⁺ current was expressed and underwent developmental changes. These events correlate well with the innervation of ectopic hair cells. New "born" hair cells at the abneural aspects of the cochlea are innervated by spiral ganglion neurons, presumably under the tropic influence of chemoattractants. The disappearance of inward currents coincides well with the attenuation of evoked electrical activity, remarkably recapitulating the development of hair cells. Ectopic hair cells underwent stepwise changes in the magnitude and kinetics of transducer currents. We propose that Atoh1 mediates trans-differentiation of morphological and functional "ancestral" hair cells that are likely to undergo diversification in an endorgan-specific manner.
Preview · Article · Mar 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Spontaneous action potentials have been described in developing sensory systems. These rhythmic activities may have instructional roles for the functional development of synaptic connections. The importance of spontaneous action potentials in the developing auditory system is underpinned by the stark correlation between the time of auditory system functional maturity, and the cessation of spontaneous action potentials. A prominent K(+) current that regulates patterning of action potentials is I(A). This current undergoes marked changes in expression during chicken hair cell development. Although the properties of I(A) are not normally classified as Ca(2+)-dependent, we demonstrate that throughout the development of chicken hair cells, I(A) is greatly reduced by acute alterations of intracellular Ca(2+). As determinants of spike timing and firing frequency, intracellular Ca(2+) buffers shift the activation and inactivation properties of the current to more positive potentials. Our findings provide evidence to demonstrate that the kinetics and functional expression of I(A) are tightly regulated by intracellular Ca(2+). Such feedback mechanism between the functional expression of I(A) and intracellular Ca(2+) may shape the activity of spontaneous action potentials, thus potentially sculpting synaptic connections in an activity-dependent manner in the developing cochlea.
[Show abstract][Hide abstract] ABSTRACT: Advances in refining the “fluid mosaic” model of the plasma membrane have revealed that it is wrought with an ordered lipid
composition that undergoes remarkable plasticity during cell development. Despite the evidence that specific signaling proteins
and ion channels gravitate toward these lipid microdomains, identification of their functional impact remains a formidable
challenge. We report that in contrast to matured auditory hair cells, depletion of membrane cholesterol in developing hair
cells produced marked potentiation of voltage-gated K+ currents (IKv). The enhanced magnitude of IKv in developing hair cells was in keeping with the reduced cholesterol-rich microdomains in matured hair cells. Remarkably,
potentiation of the cholesterol-sensitive current was sufficient to abolish spontaneous activity, a functional blueprint of
developing and regenerating hair cells. Collectively, these findings provide evidence that developmental plasticity of lipid
microdomains and the ensuing changes in K+ currents are important determinants of one of the hallmarks in the maturation of hearing.
Preview · Article · Feb 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Irreversible loss of hair cells and their innervating spiral ganglion neurons is the major reason for hearing loss. Attempts
at integrating new supplementary cell sources into the damaged inner ear have been tested extensively. This chapter reviews
the history of available cell sources and their achievements, limitations, and future developments for hearing rehabilitation.
It addressed issues regarding the “self-repair” of mammalian inner ear sensory epithelium, including (1) recruitment of the
in situ proliferation and differentiation of endogenous cells at the damaged site and (2) autologous transplantation, which
offer new optimism for helping hearing-impaired individuals.
KeywordsStem cells-Inner ear-Sensory epithelium-Rehabilitation