Voltage Sensitivity Of Channels Research Articles

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Identifying the CaV1.1 voltage-sensing domains involved in channel gating and excitation contraction coupling.

Skeletal muscle CaV1.1 is a voltage gated calcium channel, which primarily functions as voltage sensor of excitation-contraction coupling (ECC). Upon depolarization, CaV1.1 triggers the opening of the RyR1 in the sarcoplasmic reticulum, causing the calcium release event essential for skeletal muscle contraction. Upon strong stimulation, CaV1.1 elicits a calcium current with kinetics and voltage-dependence different from those of ECC. Probably, these distinct activation properties arise from the differential roles of the four CaV1.1 voltage sensing domains (VSDs). Each VSD comprises of four transmembrane helices (S1-S4); S4 segments contain 4-5 positive charges, which interact with negative countercharges in the surrounding helices. These transient ion-pair interactions facilitate and tune the S4 motion in response to changes of the membrane potential. Using structure-guided mutagenesis with patch-clamp analysis and fluorescence calcium recordings, our group identified crucial countercharges in VSD I and IV able to regulate the channel's voltage sensitivity (I and IV) and activation kinetics (IV), without interfering with ECC. However, analogous mutations in VSDs II and III showed little or no effects on either channel gaiting or ECC, corroborating the notion that the four VSDs use different molecular mechanism for regulating their activation properties. Recently, two independent voltage-clamp fluorometry studies suggested that either VSD II or III is the main activator of ECC, based on their specific kinetics and voltage sensitivity matching those of ECC. While both studies agree that perhaps only a single VSD may activate ECC, they disagree on which one that might be. Here we address this problem using site-directed mutagenesis and chimeric CaV1.1 channels expressed in dysgenic myotubes. Constructs shifting the voltage-dependence of either ECC or channel activation reveal the specific contributions of the mutated VSDs in one or the other function of CaV1.1.

Severe epilepsy phenotype with SCN1A missense variants located outside the sodium channel core region: Relationship between functional results and clinical phenotype

PurposeMost SCN1A missense variants located outside the sodium channel core region show a mild phenotype. However, there are exceptions, because of which it is challenging to determine the correlation between genotype and phenotype. In this study, we aimed to determine whether functional study could be used to determine disease severity in cases with such variants, and elucidate possible genotype-phenotype relationships. MethodsForty-seven patients with SCN1A missense variants were recruited, and one with a Dravet syndrome phenotype with an SCN1A missense variant (c.3811T>C/ p.W1271R) located outside the core region was screened with electrophysiological tests. We also reviewed functional SCN1A studies on patients with inconsistent phenotypes and genotypes, and studied the relationship between electrophysiological measurements and clinical phenotype. ResultsPatch clamp experiments showed that the W1271R variant caused significantly reduced sodium current, decreased channel voltage sensitivity, loss of channel availability, and prolonged recovery time from inactivation compared with wild type (WT), which ultimately caused a change in loss of function (LOF). Twelve cases of severe SCN1A-related epilepsy with missense variants located outside the channel core region were also included from the functional studies. Nine patients with missense SCN1A variants showed complete (3/9) or partial (6/9) physiological LOF. Two missense SCN1A variants caused physiological gain-and-loss of function (G-LOF), and one caused decreased excitability (DE). ConclusionsNot all missense variants located outside the core region cause a mild phenotype. Although current functional studies in heterologous expression systems do not accurately reflect disease severity caused by SCN1A missense variants, they could be an effective model for generation of data to study the initial effects of SCN1A missense variants.

Modulation of Voltage Sensitivity of Slow Sodium Channels by a Synthetic Cyclic Peptide

Modulation of Voltage Sensitivity of Slow Sodium Channels by a Synthetic Cyclic Peptide

Molecular mechanisms and signaling by comenic acid in nociceptive neurons influence the pathophysiology of neuropathic pain

Comenic acid (CA), a specific agonist of opioid-like receptors, effectively and safely relieves neuropathic pain by decreasing the NaV1.8 channel voltage sensitivity in the primary sensory neuron membrane. CA triggers downstream signaling cascades, in which the Na,K-ATPase/Src complex plays a key role. After leaving the complex, the signal diverges ‘tangentially’ and ‘radially’. It is directed ‘tangentially’ along the neuron membrane to NaV1.8 channels, decreasing the effective charge of their activation gating system. In the radial direction moving towards the cell genome, the signal activates the downstream signaling pathway involving PKC and ERK1/2. A remarkable feature of CA is its ability to modulate NaV1.8 channels, which relieves neuropathic pain while simultaneously stimulating neurite growth via the receptor-coupled activation of the ERK1/2-dependent signaling pathway.

Src kinase controls signaling pathways in sensory neuron triggered by low-power infrared radiation.

Low-power (non-thermal) infrared (IR) radiation with the wavelength of 10.6 μm activates the Na,K-ATPase transducer function in sensory neurons, which is manifested in decrease of NaV1.8 channel voltage sensitivity at the cellular membrane level and in inhibition of growth of chick embryo dorsal root ganglia neurites at the tissue level. It is shown that the effect of low-power IR radiation is totally blocked by a specific Src kinase inhibitor, PP2. Upon irradiation on the background of PP2, the effective charge of NaV1.8 channel activation gating system does not differ from its control value in patch-clamp experiments, and the area index of sensory ganglia neurites growth remains unchanged as compared with the control in organotypic tissue culture. The data obtained demonstrate that Src kinase is involved in intracellular signaling pathways triggered by CO2 laser low-power IR radiation by the transducer-activated mechanism. This is the first indication that in primary sensory neuron the signals of low-power IR radiation are sensed, amplified, and transduced by the Na,K-ATPase/Src complex and not by G proteins.

Voltage\xa0gating of mechanosensitive PIEZO channels

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

Inhibitory effect of the tricyclic antidepressant amitriptyline on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

Amitriptyline, a tricyclic antidepressant (TCA) drug, is widely used in treatment of psychiatric disorders. However, the side effects of amitriptyline on vascular K+ channels remain to be determined. Therefore, we investigated the effect of the tricyclic antidepressant and serotonin reuptake inhibitor amitriptyline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells, using the whole-cell patch clamp technique. The Kv current amplitudes were inhibited by amitriptyline in a concentration-dependent manner, with an apparent IC50 value of 2.2±0.14μmol/L and a Hill coefficient of 0.87±0.03. Amitriptyline shifted the activation curve to a more positive potential, but had no significant effect on the inactivation curve, suggesting that amitriptyline altered the voltage sensitivity of Kv channels. Pretreatment with Kv1.5 and Kv1.2 channel inhibitors did not alter the inhibitory effect of amitriptyline on Kv channels. Additionally, application of train pulses (1 and 2Hz) did not affect amitriptyline-induced inhibition of Kv currents, which suggested that the action of amitriptyline on Kv channels was not use (state)-dependent. From these results, we concluded that amitriptyline inhibited the channels in a concentration-dependent, but state-independent manner.

Cisapride, a selective serotonin 5-HT4-receptor agonist, inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effect of cisapride, a selective serotonin 5-HT4-receptor agonist, on voltage-dependent K+ (Kv) channels using freshly isolated smooth muscle cells from the coronary arteries of rabbits. The amplitude of Kv currents was reduced by cisapride in a concentration-dependent manner, with an IC50 value of 6.77 ± 6.01 μM and a Hill coefficient of 0.51 ± 0.18. The application of cisapride shifted the steady-state inactivation curve toward a more negative potential, but had no significant effect on the steady-state activation curve. This suggested that cisapride inhibited the Kv channel in a closed state by changing the voltage sensitivity of Kv channels. The application of another selective serotonin 5-HT4-receptor agonist, prucalopride, did not affect the basal Kv current and did not alter the inhibitory effect of cisapride on Kv channels. From these results, we concluded that cisapride inhibited vascular Kv current in a concentration-dependent manner by shifting the steady-state inactivation curve, independent of its own function as a selective serotonin 5-HT4-receptor agonist.

Y-27632, a Rho-Associated Protein Kinase Inhibitor, Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We examined the effects of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 on voltage-dependent K⁺ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Y-27632 reduced the amplitude of the Kv current in a concentration-dependent manner, with an IC₅₀ of 0.87 ± 0.06 μmol/l and a Hill coefficient of 1.48 ± 0.06. Y-27632 did not affect the steady-state activation or inactivation curves, suggesting that the drug does not affect the voltage sensitivity of Kv channels. Another ROCK inhibitor, H-1152, did not affect the Kv current and had no significant effect on the Y-27632-induced inhibition of Kv channels, indicating that the inhibitory effect of Y-27632 on the Kv current is independent of ROCK signaling. From these results, we conclude that Y-27632 inhibits the Kv channel current in a dose-dependent and ROCK signaling-independent manner.

Molecular Interactions in the Voltage Sensor Controlling Gating Properties of CaV Calcium Channels

Voltage-gated calcium channels (CaV) regulate numerous vital functions in nerve and muscle cells. To fulfill their diverse functions, the multiple members of the CaV channel family are activated over a wide range of voltages. Voltage sensing in potassium and sodium channels involves the sequential transition of positively charged amino acids across a ring of residues comprising the charge transfer center. In CaV channels, the precise molecular mechanism underlying voltage sensing remains elusive. Here we combined Rosetta structural modeling with site-directed mutagenesis to identify the molecular mechanism responsible for the specific gating properties of two CaV1.1 splice variants. Our data reveal previously unnoticed interactions of S4 arginines with an aspartate (D1196) outside the charge transfer center of the fourth voltage-sensing domain that are regulated by alternative splicing of the S3-S4 linker. These interactions facilitate the final transition into the activated state and critically determine the voltage sensitivity and current amplitude of these CaV channels.

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca2+ channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca2+ channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca2+ channels (CaV1.3 and CaV1.2) but not the N-type Ca2+ channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca2+ signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca2+ channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca2+ channel activation.

Comparative sequence analysis suggests a conserved gating mechanism for TRP channels.

The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate.

Role of the Voltage Sensing Domain S1-S4 in TRPV1 Channels

Transient Receptor Potential Vallinoid type 1 (TRPV1) channel is a voltage-, heat-, and ligand-activated, nonselective cation channel. As a member of hexahelical cation channel superfamily, TRPV1 does not feature the positively charged S4, which is responsible for the voltage sensitivity of voltage-gated ion channels (VGICs). The origin for TRPV1's voltage sensitivity must lie elsewhere. The objective of this study was to investigate the role of “classical” S1-S4 voltage sensor, in particular S4, in the TRPV1, and obtain molecular information about the functional coupling between voltage, chemical and temperature sensors. To test whether the positive charge is involved in activation and to explore the function of the S4 segment in TRPV1 activation, we made a series of single point arginine replacements that scanned residues from E536 through T556 in the S4 and characterized their response to capsaicin, protons and heat using cut-open oocyte voltage-clamp. We also constructed TRPV1/Shaker chimeras in which the S1-S4, S3-S4, or S4 segments of the TRPV1 channel were replaced by the corresponding segments of Shaker channels. The introduction of positively charged residues in the S4 segment resulted in a steeper activation curve and a defective response to capsaicin and protons. Importantly, several of the mutant channels displayed strong inhibition of hyperpolarization-activated inward currents. Three chimeras gave rise to functional channels that exhibited stronger voltage dependence at positive voltages than TRPV1. The chimeras progressively lost inward current with increasing portions replaced by the corresponding Shaker region. They were activated by protons with higher sensitivity compared with TRPV1. These results suggest that re-established outward rectification was dominant over other gating stimuli; in other words, keeping the S1-S4 in the “deactivated” state prevented activation by protons, capsaicin or temperature. In wildtype TRPV1, the S4 seems to inhibit activation by protons.

The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca2+-activated K+ (BK) Channels and Inhibits Their Activity Independent of G-protein Activation

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 μM) abolished the ANG-II (1 μM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 μM GDPβS). In BK-expressing HEK293T cells, ANG-II (1 μM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.

Palmitoylation of STREX domain confers cerebroside sensitivity to the BKCa channel

Our previous study reported that cerebrosides from traditional Chinese medicine Baifuzi directly interact with the STREX domain of BKCa channels, which in turn results in the therapeutic effect of Baifuzi on ischemic stroke. However, it is not known how cerebrosides in the plasma membrane could interact with the STREX domain that is in the cytoplasmic side. Using patch-clamp technique, effects of different cerebrosides on the BKCa channel were studied by measuring single channel currents in CHO cells expressing wild type or mutated BKCa channels. Palmitoylation of the STREX domain was removed either by site-directed mutagenesis or pharmacological inhibition. Removal of palmitoylation sites at C646 and C647 by mutating the residues to Ala abolished the ability of cerebrosides to activate the BKCa channel. In contrast, the mutation neither changed the single channel conductance nor voltage sensitivity of the channel. Both palmitoylation inhibitors tunicamycin and palmitic acid analog 2-bromopalmitate attenuated the activation of the BKCa channel by cerebrosides. Furthermore, confocal images on STREX-EGFP fragments demonstrated that STREX fragments no longer associated with the plasma membrane when the palmitoylation was removed or blocked. These findings suggest that palmitoylation of the STREX domain is necessary for cerebrosides to activate the BKCa channel and provide insight into the mechanism of how Baifuzi could exert therapeutic effect on ischemic stroke.

LRRC26 is a functional BK channel auxiliary γ subunit in arterial smooth muscle cells.

Smooth muscle cell (myocyte) large-conductance calcium (Ca)(2+)-activated potassium (BK) channels are functionally significant modulators of arterial contractility. Arterial myocytes express both pore-forming BKα and auxiliary β1 subunits, which increase channel Ca(2+) sensitivity. Recently, several leucine-rich repeat containing (LRRC) proteins have been identified as auxiliary γ subunits that elevate the voltage sensitivity of recombinant and prostate adenocarcinoma BK channels. LRRC expression and physiological functions in native cell types are unclear. Investigate the expression and physiological functions of leucine-rich repeat containing protein 26 (LRRC26) in arterial myocytes. Reverse transcription polymerase chain reaction and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence resonance energy transfer microscopy, and coimmunoprecipitation indicated that LRRC26 was located in close spatial proximity to, and associated with, plasma membrane BKα subunits. LRRC26 knockdown (RNAi) reduced total and surface LRRC26, but did not alter BKα or β1, proteins in arteries. LRRC26 knockdown did not alter Ca(2+) sparks but reduced BK channel voltage sensitivity, which decreased channel apparent Ca(2+) sensitivity and transient BK current frequency and amplitude in myocytes. LRRC26 knockdown also increased myogenic tone over a range (40-100 mm Hg) of intravascular pressures, and reduced vasoconstriction to iberiotoxin and vasodilation to NS1619, BK channel inhibitors and activators, respectively. In contrast, LRRC26 knockdown did not alter depolarization (60 mmol/L K(+))-induced vasoconstriction. LRRC26 is expressed, associates with BKα subunits, and elevates channel voltage- and apparent Ca(2+) sensitivity in arterial myocytes to induce vasodilation. This study indicates that arterial myocytes express a functional BK channel γ subunit.

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 Ca(2+)- and voltage-dependent K(+) channel (BKCa) 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 10μM AA increases the open probability (Po) of BKCa 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 BKCa 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 BKCa activation. We conclude that AA itself selectively activates the β1-associated BKCa channel, destabilizing its closed state probably by interacting with the β1-subunit, without modifying the channel voltage sensitivity. Since BKCa 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.

Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells

The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

The voltage‐dependent L‐type Ca2+ (CaV1.2) channel C‐terminus fragment is a bi‐modal vasodilator

Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.