Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP _o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

Down-regulation of the Small Conductance Calcium-activated Potassium Channels in Diabetic Mouse Atria

The small conductance Ca²⁺-activated K⁺ (SK) channels have recently been found to be expressed in the heart, and genome-wide association studies have shown that they are implicated in atrial fibrillation. Diabetes mellitus is an independent risk factor of atrial fibrillation, but the ionic mechanism underlying this relationship remains unclear. We hypothesized that SK channel function is abnormal in diabetes mellitus, leading to altered cardiac electrophysiology. We found that in streptozotocin-induced diabetic mice, the expression of SK2 and SK3 isoforms was down-regulated by 85 and 92%, respectively, whereas that of SK1 was not changed. SK currents from isolated diabetic mouse atrial myocytes were significantly reduced compared with controls. The resting potentials of isolated atrial preparations were similar between control and diabetic mice, but action potential durations were significantly prolonged in the diabetic atria. Exposure to apamin significantly prolonged action potential durations in control but not in diabetic atria. Production of reactive oxygen species was significantly increased in diabetic atria and in high glucose-cultured HL-1 cells, whereas exposure of HL-1 cells in normal glucose culture to H₂O₂ reduced the expression of SK2 and SK3. Tyrosine nitration in SK2 and SK3 was significantly increased by high glucose culture, leading to accelerated channel turnover. Treatment with Tiron prevented these changes. Our results suggest that increased oxidative stress in diabetes results in SK channel-associated electrical remodeling in diabetic atria and may promote arrhythmogenesis.     

Ca2+ Binding/Permeation via Calcium Channel,CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein,CD36, and Fatty Acid Metabolism

Ca²⁺ permeation and/or binding to the skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) facilitates activation of Ca²⁺/calmodulin kinase type II (CaMKII) and Ca²⁺ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α₁ subunit of Ca_V1.1) gene that abolishes Ca²⁺ binding within the Ca_V1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that Ca_V1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Ca_v1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized Ca_V1.1-mediated pathway that regulates energy utilization in skeletal muscle.     

Ca<Subscript>v</Subscript>1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca²⁺ channels (LTCCs, Ca_v1) open readily during membrane depolarization and allow Ca²⁺ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca²⁺-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Ca_v1.2 and Ca_v1.3), Ca_v1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Ca_v1.3^−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Ca_v1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Ca_v1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Ca_v1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.     

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.     

Surface L‐type Ca2+ channel expression levels are increased in aged hippocampus

Age‐related increase in L‐type Ca²⁺ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca²⁺ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age‐related cognitive deficits. Here, using specific antibodies against Ca_v1.2 and Ca_v1.3 subunits of LTCCs, we systematically re‐examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Ca_v1.2 and Ca_v1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Ca_v1.2 in the CA1 and CA3 regions and of Ca_v1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Ca_v1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Ca_v1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age‐related cognitive deficits cannot be attributed to a global change in L‐type channel expression nor to the level of phosphorylation of Ca_v1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age‐related increase in L‐type Ca²⁺ channel activity in CA1 pyramidal neurons.     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei

Ca_v1.2 and Ca_v1.3 are the major L-type voltage-gated Ca²⁺ channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Ca_v1.3 is essential for proper maturation. Here, we investigated the role of Ca_v1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Ca_v1.3, loss of Ca_v1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Ca_v1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Ca_v1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Ca_v1.2 and Ca_v1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.     

Differential expression of high voltage‐activated Ca2+ channel types in the rostral reticular thalamic nucleus of the absence epileptic WAG/Rij rat

In the WAG/Rij rat, a model for human absence epilepsy, spike‐wave discharges (SWD) and absence epileptic behavior develop after the age of 3 months. The rostral part of the reticular thalamic nucleus (rRTN) is involved in SWD. Ca²⁺ channels play a central role in the initiation and maintenance of burst firing activity of thalamic cells. We hypothesize that a changed expression of α₁‐subunits of one or more high voltage‐activated Ca²⁺ channel types in the rRTN underlies the development of SWD. To test this hypothesis we compared 3‐ and 6‐month‐old WAG/Rij rats with nonepileptic, age‐matched control rats. By immunocytochemistry, the expressions of α₁1.3‐, α₁2.1‐, α₁2.2‐, and α₁2.3‐subunits were shown in both strains, demonstrating the presence of Ca_v1.3, Ca_v2.1, Ca_v2.2, and Ca_v2.3 channels, respectively. Quantification of channel expression indicates that the development of SWD in WAG/Rij rats is concomitant with an increased expression of Ca_v2.1 channels in the rRTN. These channels are mainly presynaptic, as revealed by double immunofluorescence involving the presynapse marker syntaxin. The mechanism by which this increase could be related to the occurrence of SWD has been discussed. © 2004 Wiley Periodicals, Inc. J Neurobiol 58: 467–478, 2004     

Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

Caveolae position Ca_V3.2 (T‐type Ca²⁺ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca²⁺ influx to trigger Ca²⁺ sparks and large‐conductance Ca²⁺‐activated K⁺ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca²⁺ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca_v3.2 channel inhibition by Ni²⁺ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca²⁺ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca_v3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca_v1.2^?/? mice, caffeine (RyR activator) and thapsigargin (Ca²⁺ transport ATPase inhibitor), we found that sufficient SR Ca²⁺ load is a prerequisite for the Ca_V3.2‐RyR axis to generate Ca²⁺ sparks. We identified a fraction of Ca²⁺ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd³⁺ (100 µM), but insensitive to Ca_V1.2 and Ca_V3.2 channel blockade. Our data demonstrate that the VSMC Ca_V3.2‐RyR axis is down‐regulated by aging. This defective Ca_V3.2‐RyR coupling is counterbalanced by a Gd³⁺ sensitive Ca²⁺ pathway providing compensatory Ca²⁺ influx for triggering Ca²⁺ sparks in aged VSMCs.     

Store-independent Orai1-mediated Ca2+ entry and cancer

Ca²⁺ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca²⁺ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca²⁺ channels. Orai1 mediates the highly Ca²⁺-selective Ca²⁺ release-activated current (I_CRAC) and participates in the less Ca²⁺-selective store-operated current (I_SOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca²⁺ influx mechanisms, including the arachidonate-regulated Ca²⁺ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca²⁺ influx processes activated by the secretory pathway Ca²⁺-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca²⁺-activated K⁺ channel 3 (SK3) or the voltage-dependent K_v10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca²⁺ influx activation through Orai1 channels and their role in the development of different cancer features.     

A role for Ca(v)1.3 in rat intestinal calcium absorption

Active Ca²⁺ absorption through epithelial Ca²⁺ channels TRPV5/6 in duodenum is activated by hyperpolarisation. However, when diet and Ca²⁺ are plentiful, digestion products cause depolarisation. We therefore used homology-based PCR from a rat jejunal mucosal cDNA preparation to reveal the presence of the neuroendocrine L-type isoform Ca_v1.3α₁. Immunocytochemical labelling and immunoblotting localised Ca_v1.3α₁ protein in apical membrane from proximal jejunum to mid ileum. Perfusion studies in vivo with 1.25 mM luminal Ca²⁺ revealed L-type channel activity. Inhibition of glucose absorption with phloridzin strongly inhibited ⁴⁵Ca²⁺ absorption; absorption was inhibited by nifedipine and Mg²⁺ and activated by Bay K 8644, none of which affect TRPV5/6. At 10 mM Ca²⁺, nifedipine inhibited ⁴⁵Ca²⁺ absorption with a time course similar to that at 1.25 mM Ca²⁺: absorption was therefore channel-mediated rather than paracellular. We suggest that in times of dietary sufficiency, Ca_v1.3 may mediate a significant route of Ca²⁺ absorption into the body.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Ion channels in the regulation of platelet migration

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca²⁺ entry via Ca²⁺ channels. Ca²⁺ influx is sensitive to cell membrane potential which is maintained by K⁺ channel activity and/or Cl⁻ channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca²⁺ activated K⁺ channel SK4 (sk4^−/−) and their wild type littermates (sk4^+/+). According to confocal microscopy human platelets expressed the Ca²⁺ channel Orai1 and the Ca²⁺-activated K⁺ channel K_Ca3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K⁺ channel inhibitor TEA (30 mM), by SK4 specific K⁺ channel blocker clotrimazole (10 μM), but not by Cl⁻ channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4^+/+ platelets but was virtually absent in sk4^−/− platelets. In conclusion, platelet migration requires activity of the Ca²⁺ channel Orai1 and of the Ca²⁺ activated K⁺ channel SK4, but not of NPPB-sensitive Cl⁻ channels.     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.