Novel vistas of calcium-mediated signalling in the thalamus

Traditionally, the role of calcium ions (Ca²⁺) in thalamic neurons has been viewed as that of electrical charge carriers. Recent experimental findings in thalamic cells have only begun to unravel a highly complex Ca²⁺ signalling network that exploits extra- and intracellular Ca²⁺ sources. In thalamocortical relay neurons, interactions between T-type Ca²⁺ channel activation, Ca²⁺-dependent regulation of adenylyl cyclase activity and the hyperpolarization-activated cation current (I_h) regulate oscillatory burst firing during periods of sleep and generalized epilepsy, while a functional triad between Ca²⁺ influx through high-voltage-activated (most likely L-type) Ca²⁺ channels, Ca²⁺-induced Ca²⁺ release via ryanodine receptors (RyRs) and a repolarizing mechanism (possibly via K⁺ channels of the BK_Ca type) supports tonic spike firing as required during wakefulness. The mechanisms seem to be located mostly at dendritic and somatic sites, respectively. One functional compartment involving local GABAergic interneurons in certain thalamic relay nuclei is the glomerulus, in which the dendritic release of GABA is regulated by Ca²⁺ influx via canonical transient receptor potential channels (TRPC), thereby presumably enabling transmitters of extrathalamic input systems that are coupled to phospholipase C (PLC)-activating receptors to control feed-forward inhibition in the thalamus. Functional interplay between T-type Ca²⁺ channels in dendrites and the A-type K⁺ current controls burst firing, contributing to the range of oscillatory activity observed in these interneurons. GABAergic neurons in the reticular thalamic (RT) nucleus recruit a specific set of Ca²⁺-dependent mechanisms for the generation of rhythmic burst firing, of which a particular T-type Ca²⁺ channel in the dendritic membrane, the Ca²⁺-dependent activation of non-specific cation channels (I_CAN) and of K⁺ channels (SK_Ca type) are key players. Glial Ca²⁺ signalling in the thalamus appears to be a basic mechanism of the dynamic and integrated exchange of information between glial cells and neurons. The conclusion from these observations is that a localized calcium signalling network exists in all neuronal and probably also glial cell types in the thalamus and that this network is dedicated to the precise regulation of the functional mode of the thalamus during various behavioural states.     

Modulation of a Ca2+-dependent K+-current by intracellular cAMP in rat thalamocortical relay neurons

Voltage-activated calcium channels in thalamic neurons are considered important elements in the generation of thalamocortical burst firing during periods of electroencephalographic synchronization. A potent counterpart of calcium-mediated depolarization may reside in the activation of calcium-dependent potassium conductances. In the present study, thalamocortical relay cells that were acutely dissociated from the rat ventrobasal thalamic complex (VB) were studied using whole-cell patch-clamp techniques. The calcium-dependent potassium-current (I_K(Ca)) was evident as a slowly activating component of outward current sensitive to the calcium ions (Ca²⁺)-channel blocker methoxyverapamil (10 μM) and to substitution of external calcium by manganese. The I_K(Ca) was blocked by tetraethylammonium chloride (1 mM) and iberiotoxin (100 nM), but not apamin (1 μM). In addition, isolated VB neurons were immunopositive to anti-α_(913–926) antibody, a sequence-directed antibody to the α-subunit of “big” Ca²⁺-dependent K⁺-channel (BK_Ca) channels. Activators of the adenylyl cyclase cyclic adenosine monophosphate (cAMP) system, such as forskolin (20 μM), dibutyryl-cAMP (10 mM) and 3-isobutyl-1-methylxanthine (500 μM), selectively and reversibly suppressed I_K(Ca). These results suggest that a rise in intracellular cAMP level leads to a decrease in a calcium-dependent potassium conductance presumably mediated via BK_Ca type channels, thereby providing an additional mechanism by which neurotransmitter systems are able to control electrogenic activity in thalamocortical neurons and circuits during various states of electroencephalographic synchronization and de-synchronization.     

Single-nucleotide polymorphisms in vascular Ca2+-activated K+-channel genes and cardiovascular disease

In the cardiovascular system, Ca²⁺-activated K⁺-channels (KCa) are considered crucial mediators in the control of vascular tone and blood pressure by modulating the membrane potential and shaping Ca²⁺-dependent contraction. Vascular smooth muscle cells express the BK_Ca channel which fine-tunes contractility by providing a negative feedback on Ca²⁺-elevations. BK_Ca channel's ion-conducting α-subunit is encoded by the KCa1.1 gene, and the accessory and Ca²⁺-sensitivity modulating β1-subunit is encoded by the KCNMB1 gene. Vascular endothelial cells express the calmodulin-gated KCa channels IK_Ca (encoded by the KCa3.1 gene) and SK_Ca (encoded by the KCa2.3 gene). These two channels mediate endothelial hyperpolarization and initiate the endothelium-derived hyperpolarizing factor-dilator response. Considering these essential roles of KCa in arterial function, mutations in KCa genes have been suspected to contribute to cardiovascular disease in humans. So far, DNA sequence analysis in the population and patient cohorts has identified single-nucleotide polymorphisms (SNPs) in the BK_Ca β1-subunit gene as well as in the α-subunit gene (KCa1.1). Some of these SNPs produce amino acid exchanges and evoke alterations of channel functions (“gain-of-function” as well as “loss-of-function”). Moreover, the epidemiological studies showed that the presence of the E65K polymorphism in, e.g., BK_Ca β1-subunit gene (producing a “gain-of-function”) lowers the prevalence for severe hypertension and myocardial infarction. Other SNPs in the BK_Ca α-subunit gene and also in the KCa3.1 gene expressed in the endothelium have been suggested to increase the risk of cardiovascular disease. These findings from sequence analysis of human KCa genes, and epidemiological studies thus provide evidence that genetic variations and mutations in KCa channel genes contribute to human cardiovascular disease.     

Nonselective cation channels are essential for maintaining intracellular Ca2+ levels and spontaneous firing activity in the midbrain dopamine neurons

Intracellular Ca²⁺ and Ca²⁺-permeable ion channels are important in regulating the firing activity and pattern of midbrain dopamine neurons, but the role of Ca²⁺-permeable nonselective cation channels (NSCCs) on spontaneous firing activity is unclear. Therefore, we investigated how Ca²⁺-permeable NSCCs modulate spontaneous firing activity and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in acutely isolated midbrain dopamine neurons of the rat. Applications of voltage-dependent Ca²⁺ channels antagonists failed to abolish spontaneous firing activity completely, but they decreased firing rate and [Ca²⁺]_c. However, a blockade of NSCCs by 2-APB or SKF96365 more potently suppressed spontaneous firings with a depolarization of membrane potential and strong decreases in basal [Ca²⁺]_c levels. The depolarization of membrane potentials was attenuated by intracellular dialysis with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). NSCCs blockers inhibited oscillatory potentials and decreased basal [Ca²⁺]_c in the presence of tetrodotoxin. Apamin, a small-conductance Ca²⁺-activated K⁺ channel inhibitor, depolarized membrane potentials and enhanced firing rates. From these data, we conclude that NSCCs not only make up the tonic Ca²⁺ entry pathways to uphold basal [Ca²⁺]_c levels but also contribute to generation of spontaneous firings, thereby regulating spontaneous firing activities of the midbrain dopamine neurons.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Involvement of large conductance Ca2+-activated K+ channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK_Ca channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK_Ca channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK_Ca protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK_Ca in SS-induced inhibition of proliferation was examined by BK_Ca inhibition using a BK_Ca specific blocker, iberiotoxin (IBTX), and by BK_Ca transfection in BK_Ca non-expressing CHO cells. The changes in [Ca²⁺]_i were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC₄(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK_Ca channel expression. Furthermore, laminar SS induced an increase in [Ca²⁺]_i and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK_Ca channel activity on cell proliferation in response to SS was also confirmed in BK_Ca-transfected CHO cells showing a decline in proliferation. Blocking BK_Ca channel reversed its inhibitory effect, providing additional support for the involvement of BK_Ca in SS-induced proliferation reduction. Our results suggest, for the first time, that BK_Ca channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

T-type Ca2+ channels in absence epilepsy

Absence epilepsy accompanies the paroxysmal oscillations in the thalamocortical circuit referred as spike and wave discharges (SWDs). Low-threshold burst firing mediated by T-type Ca²⁺ channels highly expressed in both inhibitory thalamic reticular nuclei (TRN) and excitatory thalamocortical (TC) neurons has been correlated with the generation of SWDs. A generally accepted view has been that rhythmic burst firing mediated by T-type channels in both TRN and TC neurons are equally critical in the generation of thalamocortical oscillations during sleep rhythms and SWDs. This review examined recent studies on the T-type channels in absence epilepsy which leads to an idea that even though both TRN and TC nuclei are required for thalamocortical oscillations, the contributions of T-type channels to TRN and TC neurons are not equal in the genesis of sleep spindles and SWDs. Accumulating evidence revealed a crucial role of TC T-type channels in SWD generation. However, the role of TRN T-type channels in SWD generation remains controversial. Therefore, a deeper understanding of the functional consequences of modulating each T-type channel subtype could guide the development of therapeutic tools for absence seizures while minimizing side effects on physiological thalamocortical oscillations.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K⁺ channel have been described in vascular smooth muscle: Voltage-activated K⁺ channels (K_V) are encoded by the Kv gene family, Ca²⁺-activated K⁺ channels (BK_Ca) are encoded by the slogene, inward rectifiers (K_IR) by Kir2.0, and ATP-sensitive K⁺ channels (K_ATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4–1.6, Kv2.1, Kv9.3, Kvβ1–β4, slo α and β, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K⁺ channels are modulated by physiological vasodilators, which increase K⁺ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate K_ATP channels. These include adenosine, calcitonin gene-related peptide, and β-adrenoceptor agonists. β-adrenoceptors can also activate BK_Ca and K_V channels. Several vasoconstrictors that activate protein kinase C inhibit K_ATP channels, and inhibition of BK_Ca and K_V channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BK_Ca channels, and possibly K_ATP channels. Hypoxia leads to activation of K_ATP channels, and activation of BK_Ca channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of K_V channels. Vasodilation to increased external K⁺ involves K_IR channels. Endothelium-derived hyperpolarizing factor activates K⁺ channels that are not yet clearly defined. Such K⁺ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Ca2+ spike initiation from sensitized inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in megakaryocytes

Ca²⁺-mediated Ca²⁺ spikes were analysed in fura-2-loaded megakaryocytes. Direct Ca²⁺ loading using whole-cell dialysis induced an all-or-none Ca²⁺ spike on top of a tonic increase in cellular Ca²⁺ concentration ([Ca²⁺]_i) with a latency of 3–7 s. The latency decreased with increasingly higher concentrations of Ca²⁺ in the dialysing solution. Spike size and its initiation did not correlate with the tonic level of [Ca²⁺]_i. Thapsigargin completely abolished the Ca²⁺-induced spike initiation, suggesting that Ca²⁺ spikes originate from thapsigargin-sensitive Ca²⁺ pools. An inhibitor of phosphatidylinositide-specific phospholipase C (PLC), 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate prolonged the latency without changes of spike size in most cases (6/9 cells), but abolished the spike initiation in the other cells (3/9). The results suggest that an increase in [Ca²⁺]_i charges up the inositol-1,4,5-trisphosphate(InsP ₃)- and thapsigargin-sensitive Ca²⁺ pools which progressively sensitize to low or slightly elevated levels of InsP₃ by the action of Ca²⁺-dependent PLC until a critical Ca²⁺ content is reached, and then the Ca²⁺ spike is triggered. Thus, the limiting step of Ca²⁺ spike triggering is the initial filling process and the level of InsP₃ in megakaryocytes.     

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

Endoplasmic reticulum–mitochondria coupling: local Ca2+ signalling with functional consequences

Plasma membrane store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels are a widespread and conserved Ca²⁺ influx pathway, driving activation of a range of spatially and temporally distinct cellular responses. Although CRAC channels are activated by the loss of Ca²⁺ from the endoplasmic reticulum, their gating is regulated by mitochondria. Through their ability to buffer cytoplasmic Ca²⁺, mitochondria take up Ca²⁺ released from the endoplasmic reticulum by InsP₃ receptors, leading to more extensive store depletion and stronger activation of CRAC channels. Mitochondria also buffer Ca²⁺ that enters through CRAC channels, reducing Ca²⁺-dependent slow inactivation of the channels. In addition, depolarised mitochondria impair movement of the CRAC channel activating protein STIM1 across the endoplasmic reticulum membrane. Because they regulate CRAC channel activity, particularly Ca²⁺-dependent slow inactivation, mitochondria influence CRAC channel-driven enzyme activation, secretion and gene expression. Mitochondrial regulation of CRAC channels therefore provides an important control element to the regulation of intracellular Ca²⁺ signalling.     

Effects of aerobic exercise training on large-conductance Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

Purpose

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BK_Ca channels in rat cerebral artery.

Methods

Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside–out (I/O) patch clamp recording was performed to measure gating properties of the BK_Ca channel.

Results

Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time.

Conclusion

The results suggest that regular aerobic exercise may enhance BK_Ca channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Spontaneous Na+ and Ca2+ spike firing of cerebellar Purkinje neurons at high pressure

 The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guinea-pig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na⁺ spike parameters or the frequency of regular Na⁺ spike firing. When Na⁺ currents were blocked by 0.5–1 μM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca²⁺ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca²⁺ spike were even less prominent when K⁺ currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca²⁺ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K⁺]_o) and 1 μM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca²⁺]_o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca²⁺]_o slightly increased the firing of doublets. These results show that the single somatic Na⁺ spike is insensitive and the dendritic Ca²⁺ spike is only mildly sensitive to pressure. However, alterations in Ca²⁺ spike firing pattern suggest that modulation of dendritic K⁺ currents induce depression of dendritic excitability at pressure. Received: 19 May 1998 / Received after revision: 15 July 1998 / Accepted: 3 September 1998     

Arachidonic acid activation of BKCa (Slo1) channels associated to the β1-subunit in human vascular smooth muscle cells

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK_Ca) in vascular smooth muscle cells (VSMCs) where the presence of β₁-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10 μM AA increases the open probability (Po) of BK_Ca channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BK_Ca channel expressed in HEK cells with or without the β₁-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BK_Ca activation. We conclude that AA itself selectively activates the β₁-associated BK_Ca channel, destabilizing its closed state probably by interacting with the β₁-subunit, without modifying the channel voltage sensitivity. Since BK_Ca channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Cell membrane stretch in osteoclasts triggers a self-reinforcing Ca2+ entry pathway

Many cell types respond to mechanical membrane perturbation with intracellular Ca²⁺ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretche-voked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca²⁺ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca²⁺-activated K⁺ (K_Ca) channel activity, membrane potential changes and local and general intracellular Ca²⁺ ([Ca²⁺]_i) increases. The model we propose is that during membrane stretch, both SA channels and K_Ca channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca²⁺. During membrane stretch, Ca²⁺ influx through SA channels and hyperpolarization due to K_Ca channel activity serve as positive feedback, leading ultimately to a Ca²⁺ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca²⁺entry mechanism plays a role in regulation of osteoclast activity.