Shocking Insights for Neurovascular Coupling: Electrical Signals Ignite Calcium Dynamics in Brain Capillaries

Abstract

Brain capillaries contribute to neurovascular coupling (NVC) by sensing neural activity and coordinating upstream arteriole dilation. However, the mechanisms underlying conducted vasodilation remain incompletely understood. Recent findings (PNAS, 2024) identify a novel process, “electrocalcium coupling,” in which hyperpolarizing signals from K⁺ channels drive long-range Ca²⁺ signaling in capillaries, revealing new insights into the integration of vasodilatory signals in the brain.

Neurovascular coupling (NVC) ensures that cerebral blood flow is rapidly redirected to the brain’s most active regions [1]. This process relies on the brain’s extensive capillary network, which functions as a sensory web to detect neural activity and coordinate upstream arteriole dilation, thereby increasing local blood flow [2]. Capillary endothelial cells express multiple sensors of localized neuronal activity that orchestrate conductive vasodilation through distinct mechanisms. Reactive oxygen species generate substances that activate TRPA1 (transient receptor potential ankyrin 1) cation channels in brain capillary endothelial cells, inducing intercellular Ca²⁺ waves that are converted to electrical signals within a specialized vascular segment known as the arteriole-capillary transition (ACT) zone [3]. Prostacyclin triggers slowly propagating, Ca²⁺-dependent vasodilator signals through pathways linked to capillary endothelial cell EP1 G_q-protein coupled receptors (G_qPCRs) [4]. In contrast, stimulation of inwardly rectifying K⁺ (K_IR) channels on capillary endothelial cells with K⁺ ions generates hyperpolarizing signals that rapidly propagate through the vascular network by electrical conduction [2]. Initially, it was proposed that conducted vasodilation instigated by K_IR channels was purely electrical and independent of Ca²⁺ [2]. This assumption was challenged by a study revealing that brain capillaries exhibit diverse, neuronal activity-driven Ca²⁺ signaling patterns in vivo [5]. This activity includes long-lasting, high-amplitude events resulting from Ca²⁺ release mediated by inositol triphosphate (IP₃) receptors on the endoplasmic reticulum (ER) [5]. Intriguingly, this study also reported that blocking K_IR channels with Ba²⁺ suppressed Ca²⁺ signaling activity [5], suggesting that these events depend on plasma membrane hyperpolarization.

Building on this foundation, Nelson and colleagues recently investigated whether electrical and Ca²⁺ signals in brain capillaries operate in distinct spatiotemporal domains or are functionally linked [6]. Their findings, published in the December 2024 Proceedings of the National Academy of Sciences, provide revolutionary insight into vasodilator signal integration during NVC (Figure 1).

Figure 1. Proposed mechanism of electrocalcium coupling in brain capillaries.

Neuronally driven increases in extracellular K⁺ activating K_IR2.1 channel and/or locally released or circulating GsPCR agonists activating K_ATP channels hyperpolarize the endothelial cell plasma membrane, enhancing the electrochemical driving force for Ca²⁺ influx through TRPV4 channels. This initial Ca²⁺ signal is amplified by Ca²⁺-induced Ca²⁺ release via IP_₃ receptors (IP_₃Rs), requiring IP_₃ generated by GqPCR/phospholipase C (PLC) signaling. Depletion of ER Ca²⁺ stores may also activate store-operated Ca²⁺ entry (SOCE). Bidirectional conduction of hyperpolarization stimulates K_IR2.1 activity, regenerating the electrical signal to “pseudopropagate” Ca²⁺ signaling throughout the capillary network.

Mughal et al. used in vivo imaging and computational modeling to demonstrate that electrical signals generated by K_IR2.1 channels drive dynamic, long-distance Ca²⁺ signaling in brain capillaries by a process they call “electrocalcium coupling”. Ca²⁺ signaling activity in capillaries within the brain’s barrel cortex, measured using 2-photon microscopy, was 70–80% lower in endothelial cell-specific K_IR2.1 knockout mice compared with controls, confirming the effects of Ba²⁺ block [5]. Activation of K_IR2.1 by applying a modest concentration of KCl (10 mM) directly to a single ACT zone-adjacent capillary increased Ca²⁺ signaling activity throughout the field of view, which roughly encompasses the brain area perfused by a single parenchymal arteriole. These data suggest that hyperpolarization of the endothelial cell plasma membrane creates a propagating signal that enhances Ca²⁺ dynamics throughout the local capillary network. Further analysis showed that induced Ca²⁺ signals were evenly distributed on the arteriole and venule sides of the capillary beds, suggesting that electrical signal propagation is bidirectional. The authors propose that hyperpolarization-induced increases in the electrochemical driving force for Ca²⁺ influx, coupled with the unique properties of K_IR2.1 channels - activation by modest increases in extracellular K⁺ and membrane hyperpolarization [7] - allow rapid, long-range electrical signals to spread through the network, with hyperpolarization of endothelial cells at distal sites driving Ca²⁺ influx. The authors call this process “pseudopropagation” of Ca²⁺ signalings to distinguish it from ATP-mediated intercellular Ca²⁺ waves that travel through the brain capillary network [3].

Electrocalcium coupling in brain capillaries was further explored by inhibiting ATP-sensitive K⁺ (K_ATP) channels with the selective antagonist glibenclamide. This treatment suppressed baseline Ca²⁺ signals in brain capillary ECs, providing evidence that tonic K_ATP channel activity contributes to maintaining a hyperpolarized membrane potential. Inhibition of adenylate cyclase with SQ22536 also suppressed baseline Ca²⁺ signaling. The authors argue that this effect is due to the suppression of protein-kinase A-dependent activation of K_ATP channels downstream of an unidentified Gs-PCR (GsPCR). However, it is also possible that increased ATP levels following adenylate cyclase block account for decreased channel activity. Applying a bolus of the selective K_ATP channel activator pinacidil to a single capillary bidirectionally increased Ca²⁺ signal activity throughout the field of view, similar to the effects of K_IR2.1 channel activation. Pinacidil did not impact capillary Ca²⁺ dynamics in endothelial cell K_IR2.1 knockout mice, suggesting that membrane hyperpolarization induced by K_ATP channel activity is propagated by K_IR2.1 channels to produce long-distance Ca²⁺ signaling. Interestingly, pharmacological inhibition of TRPV4 (TRP vanilloid 4) eliminated increases in Ca²⁺ signaling activity in response to activators of K_ATP and K_IR2.1 channels, suggesting that membrane hyperpolarization drives Ca²⁺ influx through tonically activated TRPV4 channels.

Mughal et al. further identified GqPCR signaling and ER Ca²⁺ stores as essential components of electrocalcium coupling. They show endothelial cell-specific deletion of Gαq/11 expression reduces baseline capillary endothelial cell Ca²⁺ signals and prevents the induction of Ca²⁺ events in response to focal stimulation of K_ATP and K_IR2.1 channels. In addition, the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA) also significantly reduced baseline and hyperpolarization-evoked Ca²⁺ signals, underscoring the importance of intracellular Ca²⁺ stores. The authors propose that hyperpolarization-driven Ca²⁺ signals are sustained by ER Ca²⁺ release, likely mediated by IP₃ generated downstream of GqPCRs. Computational modeling studies bolstered these findings, showing that Ca²⁺ influx and ER store recharge are necessary to sustain electrocalcium coupling, suggesting a potential role for store-operated Ca²⁺ entry in this process.

The work of Mughal et al. provides compelling evidence that hyperpolarizing electrical signals generated by K⁺ channel activity are converted into Ca²⁺ signals that “pseudopropagate” over large areas of the cerebral capillary network (Figure 1). Interestingly, these findings parallel a previous study showing that intercellular Ca²⁺ signals slowly propagating through capillaries are converted into fast electrical signals within the ACT zone [3]. The dynamic interplay of electrical and Ca²⁺ signaling brain capillaries suggests a tightly regulated “exchange rate” for vasodilator mechanisms, with implications for NVC. Future research should explore the contribution of bidirectional electrocalcium coupling to CBF regulation and other critical processes, such as glymphatic clearance [8]. Understanding how electrocalcium coupling is altered in pathologies affecting NVC, such as aging, Alzheimer’s disease [9], and cerebral small vessel diseases, [10] will be crucial for identifying novel therapeutic targets.