Rat Cerebral Artery Myocytes Research Articles

Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.

Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations.

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

Role of the slo1 CRAC4 Motif in BK Channel's Ethanol Sensitivity

Cholesterol (CLR) has been recognized for its ability to interact with ion channels. Among ion channels that sense CLR presence are large conductance, voltage/Ca2+-gated K+ (BK) channels. Functional BK channels result from tetrameric association of pore-forming slo1 subunits. CLR inhibition of slo1 current may result from CLR-sensing by the slo1 cytosolic tail domain (CTD). CTD contains seven CLR-recognition amino acid consensus (CRAC) motifs that contribute to the overall CLR sensitivity of the channel, with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol (ethanol) also modifies BK channel function, this effect resulting from an orchestration of several factors that include [Ca2+]i. In native membranes, ethanol increases slo1 channel open probability (Po) at [Ca2+]i<20-30 µM whereas reduces Po at [Ca2+]i<30 µM. Both CLR-enrichment and CLR-depletion of native membranes modify ethanol action on BK channels. Thus, we set to determine the role of CLR-sensing by the CRAC4 motif in the slo1 channel's ethanol sensitivity. Slo1 cloned from rat cerebral artery myocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE:POPS (3:1 w/w) bilayers in 300/30 mM K+i/K+o gradient. At 0.3 μM Ca2+i, ethanol increased cbv1 Po by ≈3-fold. At 300 μM Ca2+i, ethanol failed to modify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR-containing (33 mol%) bilayers. The discrepancy in ethanol effect between native membranes and planar bilayers may likely arise from their difference in proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did not respond to ethanol in CLR-free bilayers, but was activated by ethanol in presence of 33 mol% CLR. Collectively, the similarity of ethanol modulation of BK channel activity studied with cbv1 vs. cbv1CRAC4T450F suggests that CRAC4 does not contribute to CLR modulation of ethanol action on slo1 at high [Ca2+]i. Support: R01AA023764 (AB), R37AA11560 (AD).

Alcohol modulation of BK channel gating depends on β subunit composition.

In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca2+i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation-inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.

Activation of Calcium- and Voltage-gated Potassium Channels of Large Conductance by Leukotriene B4

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory β1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 μm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-β2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK β1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate β1 subunit-containing channels when β1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK β1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of β1-specific BK channel activators.

BK β1 Transmembrane Regions Critically Control the Characteristic Phenotype of β1-Containing Bk Channel Currents

Voltage- and Ca2+-gated, large conductance K+ (BK) channels are ubiquitously expressed and play a key role in linking membrane excitability to Ca2+ signaling. In most tissues, BK channel complexes consist of channel-forming α and regulatory β subunits. Each β contains short intracellular N- and C-termini, and two transmembrane domains (TM1, TM2) joined by an extracellular loop. The smooth muscle-abundant BK β1 increases the channel's apparent Ca2+ sensitivity and is required for channel activation by cholane steroids (Brenner et al., 2000; Bukiya et al., 2009). The participation of different β1 regions in regulating channel function has not been fully settled. Here, we used BK channel-forming cbv1 and β1 subunits cloned from rat cerebral artery myocytes, neuron-abundant β4, and chimeras constructed by swapping regions between β1 and β4 to determine the contribution of specific β1 regions to the β1-containing BK phenotype. After co-expressing cbv1 with wt βs or chimeric βs in Xenopus oocytes, macroscopic currents were evoked by 200 ms-long, 10 mV depolarizing steps from −150 to +150 mV (VH=-80 mV). At Ca2+ levels found near the BK channel during smooth muscle cell contraction (10 μM), channel complexes that included chimeras combining both TMs from β1 with remaining regions (EC loop, IC ends) from β4 showed a phenotype (Vhalf, τact, τdeact) identical to that of channel complexes containing wt β1. However, this phenotype could not be evoked by channel complexes that included chimeras combining either β1 TM1 or β1 TM2 with the remaining β4 regions (TM2-EC loop-IC ends and TM1-EC loop-IC ends, respectively). We conclude that at physiological voltages and Ca2+, both TMs from β1 are necessary to confer the characteristic current phenotype of β1-containing BK channels. Support: R37-AA11560, R01-HL10463 (AMD).

Intracellular ATP modulates vascular distinctive P2X receptors in rat arterial smooth muscle

The extracellular nucleotide sensitive P2 receptor comprises two different groups: ionotropic P2X and metabotropic P2Y receptors. It is generally considered that the P2X receptor lacks intracellular ATP binding sites.In rat arterial myocytes, however, we identified that P2X1 and 5 receptors form a heteromultimer and found that its activation kinetics is modulated by cytosolic ATP, since a significant decrease was observed in desensitization of P2X receptor current evoked by repeated application of α,β‐methylene ATP (10μM) at 5 minute intervals with increasing intracellular ATP concentration (0, 2 and 5mM to 52.4±6.0%, 45.7±5.4% and 33.5±3.0%, respectively: n=5–13). In contrast, ADP and AMP did not modulate the current. Similar effects were observed in rat cerebral and mesenteric artery myocytes.Therefore, we performed a homology search for ATP‐binding motifs in P2X receptors, and found P2X1 and 5 receptors have the motifs at the pore‐forming transmembrane regions. In addition, we confirmed the co‐localization of P2X1 and 5 receptor proteins in each arterial tissue by RT‐PCR, western blot and immunostaining analyses.These results suggest that P2X1 and 5 receptors may play important roles in metabolic regulation in conditions such as hypoxia.

Cerebrovascular Dilation via Selective Targeting of the Cholane Steroid-Recognition Site in the BK Channel β1-Subunit by a Novel Nonsteroidal Agent

The Ca(2+)/voltage-gated K(+) large conductance (BK) channel β1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK β1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK β1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate β1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + β1) channels cloned from rat cerebral artery myocytes with a potency (EC₅₀ = 53 μM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + β2, β3, β4, or β1T169A, indicating that this drug selectively targets β1-containing BK channels via the BK β1 steroid-sensing site. HENA (3-45 μM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK β1's role in HENA action. Finally, carotid artery-infusion of HENA (45 μM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates β1-containing BK channels by targeting the steroid-sensing site in BK β1, rendering vasodilation.

Sodium 3-Hydroxyolean-12-en-30-Oate is a Novel and Selective Activator of β1 Subunit-Containing BK Channels and thus Cerebral Artery Dilator

Lithocholic acid (LCA) is a cholane steroid that causes cerebrovascular dilation via activation of β1 subunit-containing BK channels in vascular smooth muscle (Bukiya et al., 2007; 2009). In search of selective β1-containing BK activators devoid of a steroidal nucleus, we used LCA as a template and performed structure similarity search with the threshold of 70% in the Hit2lead.com database. We identified methyl 3-hydroxyolean-12-en-30-oate, which after hydrolysis, rendered sodium 3-hydroxyolean-12-en-30-oate (HENA). Patch-clamp results demonstrated that HENA activated BK channels (cbv1+β1) cloned from rat cerebral artery myocytes. While EC50 (∼50 μM), Emax (∼300 μM) and apparent Hill number (1.3) for this HENA action were undistinguishable from those of LCA, HENA's efficacy was significantly larger than that of LCA, with channel steady-state activity in presence of 150 μM HENA increasing to 200% of pre-drug values. Remarkably, HENA failed to activate recombinant, β2-, β3-, or β4-containing BK channels while activating native BK channels in rat cerebrovascular myocytes (EC50=46 μM). Therefore, HENA selectively targets β1-containing BK channels. Furthermore, HENA failed to activate cbv1+β1T169A channels, suggesting it acts via a BK β1 cholane-sensing site (Bukiya et al., 2008). HENA (3-45 μM) dilated pressurized cerebral arteries of rat and C57BL/6 mouse. Consistent with its higher efficacy on channel activity, HENA-induced dilation was significantly larger than that of LCA. As expected, genetic ablation of BK β1 suppressed HENA-induced vasodilation. Finally, closed cranial window data from anesthesized rats demonstrated that intracarotid infusion of HENA significantly dilated pial arterioles. This action was sustained in presence of 4-aminopyridine but totally prevented by paxilline, underscoring BK-mediation of HENA-induced in vivo vasodilation. This study identifies the first selective non-steroid activator of β1-containing BKs and effective cerebral artery dilator. Supported by R01-HL104631; R37-AA011560 (AMD).

The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2× increase in vascular myocyte BK activity (EC(50) = 45 μM), an action that requires β1 but not other (β2-β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK β1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit-specific pharmacology of BK channels.

The Slo1 C-Tail Domain Confers Cholesterol-Sensitivity to Arterial Smooth Muscle BK Channels

Large conductance, voltage- and calcium-gated potassium (BK) channels are known to cluster in cholesterol-rich cell membrane domains. Furthermore, cholesterol-BK channel interaction usually results in reduced channel activity (Po), as reported with native (Bolotina et al., 1989) and recombinant (Crowley et al., 2003) channels. We previously communicated that cholesterol-inhibition of BK channels cloned from rat cerebral artery myocytes (cbv1; AY330293) and reconstituted into POPE:POPS (3:1) bilayers displayed enantiospecificity and stereoselectivity, strongly suggesting that the decrease in Po involved cholesterol-recognition by a protein surface (Bukiya et al., Biophys. Soc. 2010). Using a similar system, we now demonstrate that cholesterol (16-33 mol%) inhibition of cbv1 channels is similar whether the channel is open by positive voltage or increased intracellular calcium, suggesting that the cholesterol-protein(s) interaction leads to altered channel function independently of the signal that gates the channel. Because the channel phenotype was characteristic of homomeric slo1, we hypothesize that regulation of BK gating by cholesterol involves an interaction between the steroid and the cbv1 subunit.One of the motifs that favors cholesterol-protein interactions is CRAC: -L/V-(X)(1-5)-Y-(X)(1-5)-R/K-, where X denotes any residue (Epand, 2008). We used CRAC sequence patterning and found ten CRAC motifs in cbv1, three in the core (S0-S6) and seven in the C-tail (S7-C end) domain. Thus, we next evaluated the cholesterol sensitivity of cbv1 channels truncated immediately after S6 (trS6cbv1). Remarkably, trS6cbv1 was consistently resistant to cholesterol-induced modulation (n=11) under conditions where wt cbv1 remained sensitive. Therefore, the C-tail domain confers cholesterol-sensitivity to cbv1 channels. We are currently using a combination of computational dynamics, sequential cbv1 truncation, and point mutagenesis in CRAC motifs to determine the relative contribution, if any, of these motifs to the cholesterol-sensitivity of BK channels.Support: HL104631 (AMD), UTHSC Neurosci. Postdoc. Fellowship (AKS).

Identification of Sterol-Sensing Amino Acids in Transmembrane Domain 2 of the BK Channel Beta 1 subunit

The activity of large conductance, voltage- and calcium-gated potassium (BK) channels in myocytes critically controls arterial diameter. At <300 μM in the aqueous phase (below critical micellar concentration), lithocholate (LC) and related steroids increase myocyte BK channel activity (Po) and cause arterial dilation. We showed that: 1) BK β1 TM2 is critical for the channel sensitivity to LC, and 2) BK β2-4 cannot substitute for β1 in providing LC-sensitivity to BK channels (Bukiya et al., 2008; 2009). Moreover, computational dynamics identified two LC-sensing amino acid clusters in BK β1TM2: L157,L158,T165 and T169,L172,L173. Here, we used pinpoint mutagenesis and patch-clamp electrophysiology to determine the relative contribution of each cluster to the LC-sensitivity of BK channels. BK α (cbv1) and β1 subunits cloned from rat cerebral artery myocytes (AY330293; FJ154955) were coexpressed in Xenopus oocytes, and BK currents were recorded from inside-out patches at Ca2+i=10 μM and Vm=-20 to −40mV. BK β1T165A and β1T165A,L157A,L158A mutants rendered LC-sensitive BK channels, with 150 μM LC-induced increase in Po (×1.7 times) being identical to that found with cbv1+wtβ1. In contrast, BK β1T165A,T169A and β1T169A mutants were absolutely unresponsive to ≥150 μM LC. Data unveil a critical role for β1T169 in providing LC-sensitivity to BK channels. While the β1T169S mutation was able to rescue the channel's LC-sensitivity, the β1T169S,L172A,L173A mutation failed to do so. Moreover, BK β1L172A,L173A was also LC-insensitive. These data validate our computational prediction that hydrogen bonding between T/S169 and the steroid hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for successful docking of LC onto the T169,L172,L173 cluster. Thus, T169, L172 and L173 in BK β1TM2 provide a docking surface that is essential for the LC-sensitivity of BK channels.Support: HL104631 (AMD).

Theory and applications of geometric scaling of localized calcium release events

Geometric measures of localized calcium release (LCR) events have been used to understand their biophysical basis. We found power law scaling between three such metrics-maximum amplitude (MA), mass above half-maximum amplitude (MHM), and area at half-maximum amplitude (AHM). In an effort to understand this scaling a minimal analytic model was employed to simulate LCR events recorded by confocal line scan. The distribution of logMHM as a function of logAHM, pMHM(pAHM), was dependent on model parameters such as channel open time, current size, line scan offset, and apparent diffusion coefficient. The distribution of log[MHM/AHM] as a function of logMA, p[MHM/AHM](pMA), was invariant, reflecting the gross geometry of the LCR event. The findings of the model were applied to real LCR line scan data from rabbit portal vein myocytes, rat cerebral artery myocytes, and guinea pig fundus knurled cells. pMHM(pAHM) could be used to distinguish two populations of LCR events in portal vein, even at the scale of "calcium noise," and to calculate the relative current of the two. The relative current was 2. pMHM(pAHM) could also be used to study pharmacological effects. The pMHM(pAHM) distribution of knurled cell LCR events was markedly contracted by ryanodine, suggesting a reduction in channel open time. The p[MHM/AHM](pMA) distributions were invariant across all cell types and were consistent with the model, underlying the common physical basis of their geometry. The geometric scaling of LCR events demonstrated here may help with their mechanistic characterization.

Strict Structural Requirements for Cholesterol to Inhibit BK Channels Point to Specific Steroid-Protein Interactions

Cholesterol decreases large conductance, voltage/calcium-gated potassium (BK) channel activity (NPo), an action that solely requires the channel pore-forming (α) subunit and a minimum phospholipid environment (Crowley et al., 2003). This cholesterol action is attributed to cholesterol-induced tight packing of bilayer lipids (Chang et al., 1995). Cholesterol modulation of ion channels via direct protein-steroid interactions, however, is increasingly recognized (Epshtein et al., 2009). Cholesterol analogs have been widely used to distinguish between lipid bilayer-mediated and specific protein recognition mechanisms. Thus, we probed cholesterol analogs on BK α subunits cloned from rat cerebral artery myocytes (“cbv1”; AY330293) after channel reconstitution into 3:1 (w/w) POPE:POPS bilayers. Cholesterol (33 mol%) decreased cbv1 NPo by ≈25%. In contrast, 5-cholenic acid-3β-ol, having a carboxyl group at the lateral chain end, failed to decrease NPo, underscoring the importance of a hydrophobic chain for sterol insertion into the bilayer hydrophobic core and channel inhibition. Coprostanol and cholestanol having the A/B junction in cis and trans, respectively, also decreased NPo (≤25%). In contrast, cholesterol, coprostanol and cholestanol epimers, having the C3-hydroxyl group in α-configuration, failed to decrease NPo. Therefore, a β-conformation in the hydroxyl is necessary for these monohydroxy-sterols to inhibit BK channels, strongly suggesting specific, steroid-protein interactions. Moreover, we probed the cbv1 channel with enantiomeric cholesterol (ent-cholesterol), which has physico-chemical properties similar to those of cholesterol yet can be differentially sensed by protein sites, as demonstrated by the lack of viability of C. Elegans when only ent-cholesterol is present (Crowder et al., 2001). Remarkably, ent-cholesterol repeatedly failed to reduce cbv1 NPo, buttressing the idea that cholesterol inhibition of BK channels requires steroid recognition by protein site(s), likely present in the cbv1 subunit itself.Support: AA11560 and HL077424 (AMD), UTHSC Neuroscience Postdoctoral Fellowship (AB).

Arterial Smooth Muscle BK Channel Beta1 Subunits Determine Ethanol-Induced Cerebrovascular Constriction

Ethanol at concentrations obtained in circulation after binge drinking (≤50 mM) causes cerebrovascular constriction, which primarily results from ethanol-induced inhibition of arterial smooth muscle voltage- and calcium-gated potassium (BK) channels (Liu et al., 2004). Cerebrovascular myocyte BK channels are made of channel-forming α (encoded by KCNMA1) and smooth muscle-abundant β1 subunits (encoded by KCNMB1). After cloning α (“cbv1”, AY330293) and β1 (FJ154955) subunits from rat cerebral artery myocytes, we set to identify the molecular effector of ethanol-induced inhibition of channel activity and cerebrovascular constriction. Cbv1 and cbv1+β1 channels were expressed in Xenopus oocytes and channel steady-state activity (NPo) was recorded in inside-out (I/O) macropatches at a wide Ca2+i range (0.3-100 μM). Ethanol potentiated current at Ca2+i 30 μM. Beta1 subunits shifted the crossover for ethanol-induced macroscopic current potentiation to inhibition towards lower Ca2+i (≤3 μM). This shift was paralleled by a similar shift in NPo. To evaluate β1-modulation of ethanol action in native channels, we probed ethanol on BK channels in cerebral artery myocytes isolated from wt (C57BL/6) and KCNMB1 K/O mice. Recordings were performed in I/O patches, at Vm=-20, -40mV and Ca2+i=10 μM. In myocytes from wt mice (having BK made of α and β1 subunits) 50 mM ethanol significantly decreased NPo (-23.4±9%). In contrast, ethanol reversibly increased NPo in KCNMB1 K/O myocytes. Finally, we pressurized isolated mouse cerebral arteries and evaluated the impact of β1 subunit modulation of ethanol action on organ function. In vessels from wt animals ethanol caused a robust decrease in diameter (-14%). In contrast, KCNMB1 K/O vessels were resistant to this ethanol action. Our data indicate that BK β1 subunits are the functional targets mediating ethanol-induced cerebrovascular constriction. NIH Grant AA11560 (AMD).

Essential role for inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) in UTP‐induced Ca2+ signal and diameter regulation in rat cerebral arteries

Inositol 1,4,5‐trisphosphate receptors (IP3R) regulate Ca2+ release from intracellular stores and modulate physiological functions including cell proliferation and vascular contraction. Three subtypes of IP3Rs have been described, but expression profiles and physiological functions of specific isoforms in vascular myocytes are unclear. Using RT‐PCR, immunoblotting, and immunofluorescence, we found that all three IP3R isoforms (1–3) are expressed in cerebral artery myocytes. Real‐time PCR indicated that IP3R1 was the most abundant (~82% of total), with IP3R2 and 3 less highly expressed (6 and 12%, respectively). UTP‐induced Ca2+ waves in myocytes of arterial segments were reduced from 0.7±0.1 Hz to 0±0 and 0.07± 0.04 Hz by the IP3R blockers 2‐APB (100 μM) and xestospongin C (20 μM), respectively. A shRNA suppression vector decreased IP3R1 protein by ~53% and reduced UTP‐induced Ca2+ wave frequency from 0.44±0.13 to 0.13±0.05 Hz, when compared with a scrambled control vector. In contrast, IP3R1 knockdown did not alter Ca2+ wave frequency in the absence of UTP. IP3R1 suppression also decreased UTP‐induced vasoconstriction in pressurized (20 mmHg) arteries from 30±3 μm to 15±3 μm. These results indicate that IP3R1 is the predominant IP3R isoform expressed in rat cerebral artery myocytes and is essential for UTP‐induced Ca2+ signal modification and contraction.

Abstract 1368: BK Channel β1 Subunits Are Specific Effectors Of Cholane-induced Channel Activation

The activity of large conductance, Ca 2+ - and voltage-gated potassium (BK) channels in smooth muscle critically controls vascular tone. Depolarization-induced Ca 2+ -entry in the myocyte activates BK channels, which generate outward positive current that tends to repolarize the membrane, limit Ca 2+ entry and, thus, oppose contraction. Cholane-derived steroids (e.g., lithocholic acid, LC) reduce vascular tone in isolated, resistance-size rat cerebral arteries by selective activation of myocyte BK channels. In most tissues, native BK channels consist of pore-forming α (encoded by KCNMA1 or Slo1 ) and accessory β1–4 (encoded by KCNMB1–4 ) subunits. Remarkably, KCNMB expression is tissue-specific: while KCNMB1 is highly predominant in smooth muscle, KCNMB2–4 are not. Thus, agents that target BK β1 subunits may be used to selectively modulate myocyte BK channel function. After cloning the BK α subunit from rat cerebral artery myocytes (termed “cbv1”, AY330293 ), we demonstrated that homomeric cbv1 channel steady-state activity (NPo) was not affected by acute LC application. In contrast, heteromeric cbv1+β1 channel NPo was reversibly increased by LC (+290% of control at EC max ~150 μM; EC 50 =46 μM). Whether the other BK β subunits (2–4) can substitute for β1 to evoke LC-sensitivity in the BK channel remains unknown. To test this, we applied 150 μM LC to the intracellular side of inside-out patches excised from Xenopus laevis oocytes expressing cbv1 alone or cbv1 with a given BK β subunit subtype (1–4). Currents were evoked with the membrane clamped at ±20mV and free Ca 2+ i set to 10 μM, a concentration found in the cerebral artery myocyte during contraction. As previously found, LC consistently failed to increase homomeric cbv1 NPo, while drastically enhancing heteromeric cbv1+β1 channel NPo. Remarkably, LC failed to activate cbv1+β2, cbv1+β3 and cbv1+β4 heteromeric channels. In conclusion, the BK β1 (smooth muscle-type) subunit serves as a unique sensor for cholane-derived steroids. Thus, these compounds provide a platform for designing therapeutic agents to treat cardiovascular disease where reduction of vascular tone is required.

Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (≥20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na+ and Cs+ and inhibited by Gd3+ (30 µM) and DIDS (100 µM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca2+ (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca2+-stores, first activated and then abolished SACs. Tetracaine (100 µM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca2+ and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation.