Functional roles of Cav1.3, Cav3.1 and HCN channels in automaticity of mouse atrioventricular cells

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca_v1.3, Ca_v3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca_v3.1 (Ca_v3.1^-/-), Ca_v1.3 (Ca_v1.3^-/-), channels or both (Ca_v1.3^-/-/Ca_v3.1^-/-). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca_v3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca_v3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca_v1.3 channels were required for spontaneous automaticity, because Ca_v1.3^-/- and Ca_v1.3^-/-/Ca_v3.1^-/- AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca_v1.3 channels are required for automaticity, Ca_v3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.     

Modulation of Ca(v)3.1 T-type Ca2+ channels by the ran binding protein RanBPM

Recombinant antibody-toxins are constructed via the fusion of a “carcinoma-specific” antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G₄S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]₂) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.     

Neurochemical characterization of the striatum and the nucleus accumbens in L-type Ca(v)1.3 channels knockout mice

L-type Ca(v)1.3 channels control the autonomous pacemaking of the substantia nigra (SN) dopamine (DA) neurons, which maintains the sustained release of DA in the striatum, its target structure. The persistent engagement of L-type channels during pacemaking might lead to increased vulnerability to environmental stressors or degenerative processes, providing a mechanism for the development of Parkinson's disease (PD). Interestingly, L-type channels are not necessary for pacemaking, opening the possible use of calcium channel antagonists as neuroprotective agents for PD without disturbing normal DA function. In this study we aimed to evaluate the consequences of Ca(v)1.3 channels deletion at the neurochemical level. For this purpose, tissue concentrations of DA and their respective metabolites were measured using high performance liquid chromatography (HPLC) in the striatum and the nucleus accumbens (NAcc) of mice lacking the gene for the Ca(v)1.3 channel subunit (CACNA1D) and compared to those in wild-type mice. Striatal DA level did not differ between the two groups. In contrast, the level of serotonin, glutamate, GABA, and taurine were increased by more than 50% in the striatum of Ca(v)1.3 null mice. Neurotransmitters levels in the NAcc did not differ between the different groups. In conclusion, our results neurochemically corroborate the robustness of the nigrostriatal DA neurons in the absence of Ca(v)1.3 channels, but suggest that complete deletion of this channel affected a variety of other transmitter systems.     

Dynamic redistribution of calcium sensitive potassium channels (hK(Ca)3.1) in migrating cells

Calcium‐sensitive potassium channels (K_Ca3.1) are expressed in virtually all migrating cells. Their activity is required for optimal cell migration so that their blockade leads to slowing down. K_Ca3.1 channels must be inserted into the plasma membrane in order to elicit their physiological function. However, the plasma membrane of migrating cells is subject to rapid recycling by means of endo‐ and exocytosis. Here, we focussed on the endocytic internalization and the intracellular transport of the human isoform hK_Ca3.1. A hK_Ca3.1 channel construct with an HA‐tag in the extracellularly located S3‐S4 linker was transfected into migrating transformed renal epithelial MDCK‐F cells. Channel internalization was visualized and quantified with immunofluorescence and a cell‐based ELISA. Movement of hK_Ca3.1 channel containing vesicles as well as migration of MDCK‐F cells were monitored by means of time lapse video microscopy. hK_Ca3.1 channels are endocytosed during migration. Most of the hK_Ca3.1 channel containing vesicles are moving at a speed of up to 2 µm/sec in a microtubule‐dependent manner towards the front of MDCK‐F cells. Our experiments indicate that endocytosis of hK_Ca3.1 channels is clathrin‐dependent since they colocalize with clathrin adaptor proteins and since it is impaired when a C‐terminal dileucine motif is mutated. The C‐terminal dileucine motif is also important for the subcellular localization of hK_Ca3.1 channels in migrating cells. Mutated channels are no longer concentrated at the leading edge. We therefore propose that recycling of hK_Ca3.1 channels contributes to their characteristic subcellular distribution in migrating cells. J. Cell. Physiol. 227: 686–696, 2012. © 2011 Wiley Periodicals, Inc.     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to pacemaking activity in specialized neurons and cardiac myocytes. HCN channels have a structure similar to voltage-gated K(+) channels but have a much larger putative S4 transmembrane domain and open in response to membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 are essential for gating, but not for channel folding/trafficking; 3) Arg-312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-pair with Asp residues located in the S2 and S3 domains.     

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.     

Functional interactions between voltage-gated Ca(2+) channels and Rab3-interacting molecules (RIMs): new insights into stimulus-secretion coupling

Stimulus-secretion coupling is a complex set of intracellular reactions initiated by an external stimulus that result in the release of hormones and neurotransmitters. Under physiological conditions this signaling process takes a few milliseconds, and to minimize delays cells have developed a formidable integrated network, in which the relevant molecules are tightly packed on the nanometer scale. Active zones, the sites of release, are composed of several different proteins including voltage-gated Ca(2+) (Ca(V)) channels. It is well acknowledged that hormone and neurotransmitter release is initiated by the activation of these channels located close to docked vesicles, though the mechanisms that enrich channels at release sites are largely unknown. Interestingly, Rab3 binding proteins (RIMs), a diverse multidomain family of proteins that operate as effectors of the small G protein Rab3 involved in secretory vesicle trafficking, have recently identified as binding partners of Ca(V) channels, placing both proteins in the center of an interaction network in the molecular anatomy of the active zones that influence different aspects of secretion. Here, we review recent evidences providing support for the notion that RIMs directly bind to the pore-forming and auxiliary β subunits of Ca(V) channels and with RIM-binding protein, another interactor of the channels. Through these interactions, RIMs regulate the biophysical properties of the channels and their anchoring relative to active zones, significantly influencing hormone and neurotransmitter release.     

Nitric oxide signalling augments neuronal voltage-gated L-type (Ca(v)1) and P/q-type (Ca(v)2.1) channels in the mouse medial nucleus of the trapezoid body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (Ca(V)) within this nucleus. Whole-cell patch recordings were made from brain slices from P13-15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 μM) and pharmacological blockers used to isolate Ca(2+) current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 μM) or Diethyl-ammonium-nonoate (DEA, 100 μM) were made to elucidate NO-dependent modulation of the expressed Ca(V) subtypes. A differential effect of NO on the calcium channel subtypes was observed: Ca(V)1 and Ca(V)2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (Ca(V)2.2) and R-type (Ca(V)2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36 ± 0.04 nA to 0.64 ± 0.11 nA and P/Q+R-type from 0.55 ± 0.09 nA to 0.94 ± 0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways.     

Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Ca_v1.3 Ca²⁺ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Ca_v1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Ca_v1.3 properties may support distinct roles of Ca_v1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Ca_v1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Ca_v1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Ca_v1.3 channels in mouse IHCs. In mature IHCs, Ca_v1.3 channels activate more rapidly and exhibit greater Ca²⁺-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Ca_v1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca²⁺ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca²⁺ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca²⁺, which restricts negative feedback regulation of Ca_v1.3 channels to incoming Ca²⁺ ions in mature IHCs. Together with faster Ca_v1.3 activation kinetics, increased reliance of Ca_v1.3 CDI on local Ca²⁺ may sharpen presynaptic Ca²⁺ signals and improve temporal aspects of sound coding in mature IHCs.     

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca_vα₁ subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca_vα₁ subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity”.     

Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear

The hyperpolarization-activated current, I_h, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. I_h has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry I_h in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to I_h in SGNs. Deletion of Hcn1 resulted in reduced conductance (G_h), slower activation kinetics (τ_fast), and hyperpolarized half-activation (V_1/2) potentials. We demonstrate that I_h contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated I_h is critical for synchronized spike timing in SGNs. Together, our data indicate that I_h contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.     

Fibroblast growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment epithelial cells act via stimulation of neuroendocrine L-type channels (Ca(v)1.3).

In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba(2+) currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10(-5) M) but was not influenced by the FGFR1 blocker SU5402 (2 x 10(-5) M) or by the blocker for src-kinase herbimycin A (10(-5) M). Immunoprecipitation of FGFR2 led to coprecipitation of alpha 1D Ca(2+) channel subunits and precipitation of alpha 1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with alpha 1D Ca(2+) channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The alpha 1D subunit-specific band were stained with antiphosphotyrosine antibodies. We conclude that FGFR2 acts via a different signaling cascade than FGFR1. This cascade involves an src-kinase-independent, close functional interaction of FGFR2 and the alpha subunit of neuroendocrine L-type channels.     

Modulation of Ca(v)1 and Ca(v)2.2 channels induced by nitric oxide via cGMP-dependent protein kinase

The unconventional gaseous transmitter nitric oxide (NO) markedly influences most of mechanisms involved in the regulation of intracellular Ca2+ homeostasis. In excitable cells, Ca2+ signaling mainly depends on the activity of voltage-gated Ca2+ channels (VGCCs). In the present paper, we will review data from our laboratory and others characterizing NO-induced modulation of Ca(v)1 (L-type) and Ca(v)2.2 (N-type) channels. In particular, we will explore experimental evidence indicating that NO's inhibition of channel gating is produced via cGMP-dependent protein kinase and examine some of the numerous cell functions that are potentially influenced by the action of NO on Ca2+ channels.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.