Myoung Kyu Park

Perinuclear, perigranular and sub‐plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport

We have identified three distinct groups of mitochondria in normal living pancreatic acinar cells, located (i) in the peripheral basolateral region close to the plasma membrane, (ii) around the nucleus and (iii) in the periphery of the granular region separating the granules from the basolateral area. Three‐dimensional reconstruction of confocal slices showed that the perigranular mitochondria form a barrier surrounding the whole of the granular region. Cytosolic Ca2+ oscillations initiated in the granular area triggered mitochondrial Ca2+ uptake mainly in the perigranular area. The most intensive uptake occurred in the mitochondria close to the apical plasma membrane. Store‐operated Ca2+ influx through the basolateral membrane caused preferential Ca2+ uptake into sub‐plasmalemmal mitochondria. The perinuclear mitochondria were activated specifically by local uncaging of Ca2+ in the nucleus. These mitochondria could isolate nuclear and cytosolic Ca2+ signalling. Photobleaching experiments indicated that different groups of mitochondria were not luminally connected. The three mitochondrial groups are activated independently by specific spatiotemporal patterns of cytosolic Ca2+ signals and can therefore participate in the local regulation of Ca2+ homeostasis and energy supply.

The endoplasmic reticulum as one continuous Ca2+ pool: visualization of rapid Ca2+ movements and equilibration

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca2+ store or is composed of separate subunits by monitoring movements of Ca2+ and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca2+ and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca2+ store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca2+ concentration throughout the cell, but repetitive cytosolic Ca2+ spikes, induced by a low ACh concentration, hardly reduced the ER Ca2+ level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca2+ liberation. Ca2+ released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.

The endoplasmic reticulum: one continuous or several separate Ca2+ stores?

The Ca2+ store and sink in the endoplasmic reticulum (ER) is important for Ca2+ signal integration and for conveyance of information in spatial and temporal domains. Textbooks regard the ER as one continuous network, but biochemical and biophysical studies revealed apparently discrete ER Ca2+ stores. Recent direct studies of ER lumenal Ca2+ movements show that this organelle system is one continuous Ca2+ store, which can function as a Ca2+ tunnel. The concept of a fully connected ER network is entirely compatible with evidence indicating that the distribution of Ca2+ -release channels in the ER membrane is discontinuous with clustering in certain localities.

Local uncaging of caged Ca2+ reveals distribution of Ca2+-activated Cl− channels in pancreatic acinar cells

In exocrine acinar cells, Ca2+-activated Cl− channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl− uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca2+, was used to reveal the Cl− channel distribution in mouse pancreatic acinar cells, where ≈90% of the current activated by Ca2+ in response to acetylcholine was carried by Cl−. When caged Ca2+ in the cytosol was uncaged locally in the apical pole, the Cl− current was activated, whereas local Ca2+ uncaging in the basal or lateral areas of the cell had no effect. Even when Ca2+ was uncaged along the whole inner surface of the basolateral membrane, no Cl− current was elicited. There was little current deactivation at a high cytosolic Ca2+ concentration ([Ca2+]c), but at a low [Ca2+]c there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca2+-activated Cl− channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca2+]c and membrane potential. Ca2+-activated Cl− channels do not mediate Cl− uptake at the base, but acetylcholine-elicited local [Ca2+]c spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.

Contribution of Ca2+‐activated K+ channels and non‐selective cation channels to membrane potential of pulmonary arterial smooth muscle cells of the rabbit

1 Using the perforated patch‐clamp or whole‐cell clamp technique, we investigated the contribution of Ca2+‐activated K+ current (IK(Ca)) and non‐selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2 The resting membrane potential (Vm) was ‐39·2 ± 0·9 mV (n= 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5·6 ± 1·1 Hz and ‐7·7 ± 0·7 mV (n= 12), respectively. In the voltage‐clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3 Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4 Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5 The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1·7 : 1·3 : 1 : 0·9, indicating that it is a non‐selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be ‐13·9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6 From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+‐activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

Different modulation of Ca-activated K channels by the intracellular redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit.

AstractWe investigated the electrical responses of Ca-activated K (KCa) currents induced by hypoxia and reduction or oxidation of the channel protein in pulmonary (PASMC) and ear (EASMC) arterial smooth muscle cells using the patch-clamp technique. In cell-attached patches, in the presence of a high K solution (containing 0.316 (μM Ca2+), the activity of KCa channels from PASMC was decreased (by 49±7% compared to control, pipette potential = −70 mV) by changing to a hypoxic solution (1 mM Na2S2O4, aeration with 100% N2 gas). EASMC channels did not respond to hypoxia. In order to investigate the possible mechanisms involved, using inside-out patches bathed symmetrically in 150 mM KCl, we applied redox couples to the intracellular side. Reducing agents, such as dithiothreitol (DDT, 5 mM), reduced glutathione, (GSH, 5 mM), and nicotinamide adenine dinucleotide reduced (NADH, 2 mM) decreased PASMC, but not EASMC, KCa channel activity. However, oxidizing agents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM), oxidized glutathione (GSSG, 5 mM) and NAD (2 mM) increased KCa channel activity in both PASMC and EASMC. The increased activity due to oxidizing agents was restored by applying reducing agents. From these results, we could suggest that the basal redox state of the EASMC KCa channel is more reduced than that of the PASMC channel, since the response of KCa channels of the EASMC to intracellular reducing agents differs from that of the PASMC. This difference may be related to the different responses of PASMC and EASMC KCa channels to hypoxia.

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.

Regional Interaction of Endoplasmic Reticulum Ca2+ Signals between Soma and Dendrites through Rapid Luminal Ca2+ Diffusion

The endoplasmic reticulum (ER) Ca2+ store plays a key role in integration and conveyance of Ca2+ signals in highly polarized neurons. The interconnected ER network in neurons generates Ca2+ signals in local domains, but the regional interaction is unclear. Here, we show that continuous or repetitive applications of caffeine produced robust Ca2+ release from the ER Ca2+ store in dendritic areas without severe store depletion, but that similar stimuli applied to soma caused rapid store depletion in acutely isolated midbrain dopamine neurons. Partial emptying of the ER Ca2+ store within a dendrite caused a similar level of store depletion in unstimulated dendrites, as well as in soma. Photobleaching and local stimulation experiments revealed that Ca2+ and the dye trapped within the ER diffused rapidly from the soma to dendrites up to 90 μm, which we could resolve, suggesting that the ER network acts as a functional tunnel for rapid Ca2+ transport. These data imply that the ER in soma acts as a Ca2+ reservoir supplying Ca2+ to the dendritic store, and that the dendritic store, hence, is able to respond to Ca2+-mobilizing input signals endurably.

Modulation of voltage-dependent K+ channel by redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

Abstract It has been suggested that hypoxic pulmonary vasoconstriction (HPV) results from the depolarization that is induced by the suppression of K+ current in pulmonary arterial smooth muscle cells (PASMC). We tested the hypothesis that the effect of the cellular redox potential on voltage-sensitive K+ (Kv) current is involved in HPV as a primary sensing mechanism. Kv current in PASMC and ear arterial smooth muscle cells (EASMC) of the rabbit was recorded using the whole-cell patch-clamp technique, and the effect of redox agents [dithiothreitol, DTT and 2,2’-dithio-bis(5-nitropyridine), DTBNP] was tested. Kv current was decreased by DTT, but increased by DTBNP. DTT accelerated the inactivation kinetics, but did not affect steady-state activation and inactivation, whereas DTBNP accelerated activation kinetics. Kv current has a non-inactivating window in the range of from –40 mV to +10 mV. The resting membrane potential measured using the nystatin-perforated-patch method, however, lay between –50 mV and –30 mV and was not depolarized by 5 mM 4-aminopyridine. The membrane-impermeable oxidizing agent DTNB has no effect on Kv current, suggesting that redox modulation sites are intracellular sulphydryl groups. In EASMC, Kv current was decreased by DTT, but increased by DTBNP, indicating that the redox-potential-induced modulation of Kv current in EASMC and in PASMC is the same. It is therefore concluded that Kv current is modulated by the cellular redox potential, but that this modulation is not involved in HPV as a primary sensing mechanism.

The Endoplasmic Reticulum as an Integrator of Multiple Dendritic Events

Dendrites are integrating elements that receive numerous subsets of heterogeneous synaptic inputs, which generate temporally and spatially distinct changes in membrane potential and intracellular Ca2+ levels in local domains. The ubiquitously distributed endoplasmic reticulum (ER) in dendrites is luminally connected to the bulk ER in the soma, constituting a huge interconnected intracellular network that allows rapid Ca2+ diffusion and equilibration. The ER is an excitable organelle that can elicit or terminate cytosolic Ca2+ signals in local or global domains. The absolute level or changes in the Ca2+ concentration in the ER lumen are also very important for the synthesis and maturation of proteins, regulation of gene expression, mitochondrial functions, neuronal excitability, and synaptic plasticity. Through the connected lumen of the ER, information from multiple dendritic events in neurons appears to be delivered into the bulk ER in the soma. Therefore, the ER network in neurons is emerging as a conveyor and integrator of signals. In this article, we will discuss the various roles of the ER and the functional and structural organization of the ER network in neurons. NEUROSCIENTIST 14(1):68—77, 2008.