Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca²⁺ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca²⁺]_i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca²⁺. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca²⁺ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca²⁺ extrusion with a high concentration of Gd³⁺, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca²⁺]_i oscillations, and this was reversed by use of high concentrations of Gd³⁺; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca²⁺ entry in HEK293 cells. These findings indicate that the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in HEK293 cells depends upon the Ca²⁺ sensor, Stim1, and calcium release-activated Ca²⁺ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.     

Structurally delineating stromal interaction molecules as the endoplasmic reticulum calcium sensors and regulators of calcium release-activated calcium entry

Summary:  The endoplasmic reticulum (ER) lumen stores a crucial source of calcium (Ca²⁺) maintained orders of magnitude higher than the cytosol for the activation of a plethora of cellular responses transmitted in health and disease by a mutually efficient and communicative exchange of Ca²⁺ between compartments. A coordination of the Ca²⁺ signal is evident in the development of Ca²⁺ release-activated Ca²⁺ (CRAC) entry, vital to lymphocyte activation and replenishing of the ER Ca²⁺ stores, where modest decreases in ER luminal Ca²⁺ induce sustained increases in cytosolic Ca²⁺ sourced from steadfast extracellular Ca²⁺ supplies. While protein sensors that transduce Ca²⁺ signals in the cytosol such as calmodulin are succinctly understood, comparative data on the ER luminal Ca²⁺ sensors is only recently coming to light with the discovery that stromal interaction molecules (STIMs) sense variations in ER stored Ca²⁺ levels in the functional regulation of plasma membrane Orai proteins, the major component of CRAC channel pores. Drawing from data on the role of STIMs in the modulation of CRAC entry, this review illustrates the structural features that delimit the functional characteristics of ER Ca²⁺ sensors relative to well known cytoplasmic Ca²⁺ sensors.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.     

The actin cytoskeleton in store-mediated calcium entry

Store-mediated Ca²⁺ entry is the main pathway for Ca²⁺ influx in platelets and many other cells. Several hypotheses have considered both direct and indirect coupling mechanisms between the endoplasmic reticulum and the plasma membrane. Here we pay particular attention to new insights into the regulation of store-mediated Ca²⁺ entry: the role of the cytoskeleton in a secretion-like coupling model. In this model, Ca²⁺ entry may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, that shows close parallels to the events mediating secretion. As with secretion, the actin cytoskeleton plays an inhibitory role in the activation of Ca²⁺ entry by preventing the approach and coupling of the endoplasmic reticulum with the plasma membrane, making cytoskeletal remodelling a key event in the activation of Ca²⁺ entry. We also review recent advances investigating the regulation of store-mediated Ca²⁺ entry by small GTPases and phosphoinositides, which might be involved in the store-mediated Ca²⁺ entry pathway through roles in the remodelling of the cytoskeleton.     

Capacitative calcium entry: from concept to molecules

Summary:  Rapid to moderately rapid changes in intracellular Ca²⁺ concentration, or Ca²⁺ signals, control a variety of critical cellular functions in the immune system. These signals are comprised of Ca²⁺ release from intracellular stores coordinated with Ca²⁺ influx across the plasma membrane. The most common mechanisms by which these two modes of signaling occur is through inositol 1,4,5-trisphosphate (IP₃)-induced release of Ca²⁺ from the endoplasmic reticulum (ER) and store-operated Ca²⁺ entry across the plasma membrane. The latter process was postulated over 20 years ago, and in just the past few years, the key molecular players have been discovered: STIM proteins serve as sensors of Ca²⁺ within the ER which communicate with and activate plasma membrane store-operated channels composed of Orai subunits. The process of store-operated Ca²⁺ entry provides support for oscillating Ca²⁺ signals from the ER and also provides direct activator Ca²⁺ that signals to a variety of downstream effectors.     

Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries

The present study was aimed at investigating whether, besides its pivotal role in Ca²⁺-independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca²⁺ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 μM) completely relaxed the contraction evoked by noradrenaline (1 μM) and simultaneously inhibited the Ca²⁺ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF₄⁻, while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca²⁺. The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31-8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K⁺ channel blockers or with the Ca²⁺ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca²⁺ from non-mitochondrial stores evoked by Ins P ₃ in permeabilised aortic cells, or the Ca²⁺ signals evoked by thapsigargin or caffeine. The capacitative Ca²⁺ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba²⁺ activated by noradrenaline in the presence of nimodipine was blocked by 10 μM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca²⁺ entry distinct from voltage- or store-operated channels in rat arteries.     

Mitochondria as all-round players of the calcium game

Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca²⁺ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca²⁺, the possibility that physiological stimuli that raise the [Ca²⁺] of the cytoplasm could trigger major mitochondrial Ca²⁺ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca²⁺] rises. The direct measurement of mitochondrial [Ca²⁺] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca²⁺ signal is always paralleled by a much larger rise in [Ca²⁺] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca²⁺ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca²⁺ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca²⁺ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca²⁺ sources; (iii) the physiological consequences of Ca²⁺ transport for both mitochondrial function and the modulation of the cytoplasmic Ca²⁺ signal; and (iv) evidence that alterations of mitochondrial Ca²⁺ signalling may occur in pathophysiological conditions.     

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca²⁺ stores activates influx of extracellular Ca²⁺ through 'store-operated' Ca²⁺ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca²⁺ stores. Here, using high resolution imaging with the membrane-associated Ca²⁺ indicator, FFP-18, it is shown that store-operated Ca²⁺ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca²⁺, depletion of ER Ca²⁺ stores using cyclopiazonic acid, an ER Ca²⁺-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca²⁺ causes localized Ca²⁺ influx that first increases [Ca²⁺]_SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs , markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca²⁺ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca²⁺ stores.     

mGluR1/5 subtype-specific calcium signalling and induction of long-term potentiation in rat hippocampal oriens/alveus interneurones

Hippocampal inhibitory interneurones demonstrate pathway- and synapse-specific rules of transmission and plasticity, which are key determinants of their role in controlling pyramidal cell excitability. Mechanisms underlying long-term changes at interneurone excitatory synapses, despite their importance, remain largely unknown. We use two-photon calcium imaging and whole-cell recordings to determine the Ca²⁺ signalling mechanisms linked specifically to group I metabotropic glutamate receptors (mGluR1α and mGluR5) and their role in hebbian long-term potentiation (LTP) in oriens/alveus (O/A) interneurones. We demonstrate that mGluR1α activation elicits dendritic Ca²⁺ signals resulting from Ca²⁺ influx via transient receptor potential (TRP) channels and Ca²⁺ release from intracellular stores. By contrast, mGluR5 activation produces dendritic Ca²⁺ transients mediated exclusively by intracellular Ca²⁺ release. Using Western blot analysis and immunocytochemistry, we show mGluR1α-specific extracellular signal-regulated kinase (ERK1/2) activation via Src in CA1 hippocampus and, in particular, in O/A interneurones. Moreover, we find that mGluR1α/TRP Ca²⁺ signals in interneurone dendrites are dependent on activation of the Src/ERK cascade. Finally, this mGluR1α-specific Ca²⁺ signalling controls LTP at interneurone synapses since blocking either TRP channels or Src/ERK and intracellular Ca²⁺ release prevents LTP induction. Thus, our findings uncover a novel molecular mechanism of interneurone-specific Ca²⁺ signalling, critical in regulating synaptic excitability in hippocampal networks.     

Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach

The pathways involved in Ca²⁺ signalling in the uterus remain incompletely understood, impairing our ability to prevent preterm and difficult labours. In this review we focus on two elements in the pathway of Ca²⁺ signalling that have recently emerged as playing important roles: membrane lipid rafts and the sarcoplasmic reticulum. We examine the evidence for lipid rafts in the uterus and discuss their functional role. We suggest that the increases in cytosolic [Ca²⁺] and contractility that occur with raft disruption are due, at least in part, to effects on large conductance Ca²⁺-activated K⁺ (BK) channels that are localized to rafts. The role of the SR in contributing to subsarcolemmal cytosolic microdomains in uterus is evaluated, along with its interactions with ion channels on the plasma membrane. Thus, signalling microdomains play an important, but incompletely understood, role in the uterus, and integrating them into other Ca²⁺ signalling pathways is a challenge for further research. We suggest that the role of the SR changes in pregnancy, from promoting quiescence via BK channels or SR Ca²⁺ uptake, to promoting Ca²⁺ entry and contractility at term, and relate data on lipid rafts to clinical outcome in obese pregnant women.     

Intracellular calcium signalling in magnocellular neurones of the rat supraoptic nucleus: understanding the autoregulatory mechanisms

Oxytocin and vasopressin, released at the soma and dendrites of neurones, bind to specific autoreceptors and induce an increase in [Ca²⁺]₁. In oxytocin cells, the increase results from a mobilisation of Ca²⁺ from intracellular stores, whereas in vasopressin cells, it results mainly from an influx of Ca²⁺ through voltage-dependent channels. The response to vasopressin is coupled to phospholipase C and adenylyl-cyclase pathways which are activated by V₁ (V_1a and V_1b)- and V₂-type receptors respectively. Measurements of [Ca²⁺]₁ in response to V_1a and V₂ agonists and antagonists suggest the functional expression of these two types of receptors in vasopressin neurones. The intracellular mechanisms involved are similar to those observed for the action of the pituitary adenylyl-cyclase-activating peptide (PACAP). Isolated vasopressin neurones exhibit spontaneous [Ca²⁺]₁ oscillations and these are synchronised with phasic bursts of electrical activity. Vasopressin modulates these spontaneous [Ca²⁺]₁ oscillations in a manner that depends on the initial state of the neurone, and such varied effects of vasopressin may be related to those observed on the electrical activity of vasopressin neurones in vivo.     

Parameters of calcium homeostasis in normal neuronal ageing

The last decade has witnessed a significant turn in our understanding of the mechanisms responsible for the decline of cognitive functions in aged brain. As has been demonstrated by detailed morphological reassessments, the senescence-related changes in cognition cannot be attributed to a simple decrease in the number of neurons. It is becoming clearer that a major cause of age-induced deterioration of brain capability involves much subtler changes at the level of synapses. These changes are either morphological, i.e. reduction in the number of effective synapses and/or functional alterations, i.e. changes in the efficacy of remaining synapses. Important questions are now raised regarding the mechanisms which mediate these synaptic changes. Clearly, an important candidate is calcium, the cytotoxic role of which is already firmly established. The wealth of evidence collected so far regarding the changes of Ca²⁺ homeostasis in aged neurons shows that the overall duration of cytoplasmic Ca²⁺ signals becomes longer. This is the most consistent result, demonstrated on different preparations and using different techniques. What is not yet clear is the underlying mechanism, as this result could be explained either through an increased Ca²⁺ influx or because of a deficit in the Ca²⁺ buffering/clearance systems. It is conceivable that these prolonged Ca²⁺ signals may exert a local excitotoxic effect, removing preferentially the most active synapses. Uncovering of the role of Ca²⁺ in the synaptic function of the aged brain presents an exciting challenge for all those involved in the neurobiology of the senescent CNS.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

Roles of Cav channels and AHNAK1 in T cells: the beauty and the beast

Summary:  T lymphocytes require Ca²⁺ entry though the plasma membrane for their activation and function. Recently, several routes for Ca²⁺ entry through the T-cell plasma membrane after activation have been described. These include calcium release-activated channels (CRAC), transient receptor potential (TRP) channels, and inositol-1,4,5-trisphosphate receptors (IP₃Rs). Herein we review the emergence of a fourth new route for Ca²⁺ entry, composed of Ca_v channels (also known as L-type voltage-gated calcium channels) and the scaffold protein AHNAK1 (AHNAK/desmoyokin). Both helper (CD4⁺) and killer (CD8⁺) T cells express high levels of Ca_v1 α1 subunits (α1S, α1C, α1D, and α1F) and AHNAK1 after their differentiation and require these molecules for Ca²⁺ entry during an immune response. In this article, we describe the observations and open questions that ultimately suggest the involvement of multiple consecutive routes for Ca²⁺ entry into lymphocytes, one of which may be mediated by Ca_v channels and AHNAK1.     

Checking your SOCCs and feet: the molecular mechanisms of Ca2+ entry in skeletal muscle

It has long been known that skeletal muscle contraction persists in the absence of extracellular Ca²⁺. Nevertheless, recent evidence indicates that multiple distinct Ca²⁺ entry pathways exist in skeletal muscle: one active at negative potentials that requires store depletion (store-operated calcium entry or SOCE) and a second that is independent of store depletion and is activated by depolarization (excitation-coupled calcium entry or ECCE). This review highlights recent findings regarding the molecular identity, subcellular localization, and inter-relationship between SOCE and ECCE in skeletal muscle. The respective roles of ryanodine receptors (RyRs), dihydropyridine receptors (DHPRs), inositol-1,4,5-trisphosphate receptors (IP₃Rs), canonical transient receptor potential channels (TRPCs), STIM1 Ca²⁺ sensor proteins, and Orai1 Ca²⁺ permeable channels in mediating SOCE and ECCE in skeletal muscle are discussed. Differences between SOCE and ECCE in skeletal muscle with Ca²⁺ entry mechanisms in non-excitable cells are also reviewed. Finally, potential physiological roles for SOCE and ECCE in skeletal muscle development and function, as well as other currently unanswered questions and controversies in the field are also considered.     

Dendritic cell altered states: what role for calcium?

Summary:  Ca²⁺-driven responses in dendritic cells (DCs) are less well characterized than in lymphocytes. When DCs undergo a sequence of activation/maturation events, typically beginning with exposure to pathogens in the periphery, Ca²⁺ entry into the cytosol from stores in the endoplasmic reticulum or from outside the cell can occur at various steps and participate in intracellular signaling. However, not all cellular processes identified in these cells are Ca²⁺ dependent. While immigration of precursor DCs into the peripheral tissues as well as emigration to secondary lymphoid sites following microbial challenge depend on processes that involve Ca²⁺, other processes such as DC maturation in response to Toll-like receptor agonist stimulation appear not to. Certain microbial stimuli and host-derived chemokines induce Ca²⁺ entry that is important for the induced responses. In this article, we review the current state of our understanding of the role of Ca²⁺ in DC biology and argue that homeostatic control of Ca²⁺ levels in these cells is critical for maintaining their proper function. We also consider evidence for intercellular transmission of Ca²⁺ signals between DCs that are physically linked by thin membranous extensions termed tunneling nanotubules.     

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca²⁺ buffering capacity and an exceptional vulnerability to Ca²⁺-mediated excitotoxic events. In this report, we investigated local Ca²⁺ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca²⁺ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca²⁺-activated, apamin-sensitive K⁺ currents ( I _KCa), demonstrating a functional link between Ca²⁺ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently I _KCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca²⁺ release profiles in HMNs, where low buffering capacities enhance the role of Ca²⁺-regulating organelles as local determinants of [Ca²⁺]_i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.