Rationale T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.
Ca sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca entry. Beginning with Ca 3.2 channel inhibition, Ni was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a Ca 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca channels. Furthermore, computational and experimental observations illustrated that Ca spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of Ca 1.2 and Ca 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na /Ca exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca spark/STOC production and thus precisely tune negative electrical feedback.
CaV1.2 (L‐type) along CaV3.1/CaV3.2 (T‐type) are the principal subtypes of voltage‐gated Ca2+ channels (VGCC) expressed in cerebral arterial smooth muscle. While studies have long discerned the functional role of CaV1.2, the physiological significance of CaV3.x expression is uncertain. Recent immunohistochemical analysis noted that CaV3.2 localizes in close proximity to ryanodine receptors (RyR) on the sarcoplasmic reticulum. From these observations, we hypothesized that CaV3.2 triggers Ca2+ ‐induced Ca2+ release (RyR), activating large‐conductance Ca2+‐activated K+ channels (BKCa) to attenuate arterial constriction. Structural analysis involving immuno‐labeling approaches, electron microscopy, and 3D‐tomomography revealed a microdomain structure in cerebral arteries comprised of CaV3.2 and RyR. Using mathematical techniques, a microdomain model was subsequently developed and it revealed that Cav3.2 was capable of activating RyR and induce repetitive CICR‐like events. In keeping with these theoretical observations, perforated patch clamp electrophysiology revealed that Ni2+ (50 μM, Cav3.2 inhibitor) attenuated the frequency and amplitude of BKCa‐mediated spontaneous transient outward currents (STOCs). Pressurized cerebral arteries were also shown to depolarized and constricted to micromolar Ni2+. The magnitude of these functional responses was comparable to paxilline, a BKCa channel inhibitor. In summary, findings indicate for the first time that CaV3.2 channels are capable of driving a CICR‐like process that moderates arterial constriction through a feedback response involving BKCa channels.
Recent work has revealed a microdomain in cerebral arterial smooth muscle comprised of caveoli and sarcoplasmic reticulum. Immunolabeling techniques indicate that T‐type Ca2+ channels and ryanodine receptors localize to this microdomain while L‐type Ca2+ channels do not. Given these observations, T‐type Ca2+ channels were hypothesized to initiate a CICR‐like response. To address this hypothesis, a model of smooth muscle Ca2+ handling was developed. Our physical model was derived from direct dimensional measurements and consisted of a large cytosolic domain and a structured subspace of caveoli and SR. T‐type Ca2+ channels and ryanodine receptors were localized to the subspace whereas L‐type Ca2+ channels, Na+/Ca2+ exchanger, PMCA and SERCA pump were placed outside this region. The kinetics of SR Ca2+ uptake/release were derived from previous models. The activity of sacrolemmal pumps, exchanges and channels were obtained from various sources and scaled in a smooth muscle specific manner. A 20 mV depolarizing step from −60 mV induced an initial CICR process that gave way to periodic SR Ca2+ release every 10sec. This spark‐like release pattern was dependent upon continuous Ca2+ influx through T‐type Ca2+ channels and the recovery of ryanodine receptors from Ca2+‐induced inactivation. Varying the depolarizing step from −50 to −30 mV did not affect the frequency of SR release, a finding consist with T‐type Ca2+ channels acting as a two‐state switch. Supported by the Canadian Institute for Health Research.
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