BK Β1 Research Articles

Functional effects of KCNQ K+ channels in airway smooth muscle

KCNQ (Kv7) channels underlie a voltage-gated K+ current best known for control of neuronal excitability, and its inhibition by Gq/11-coupled, muscarinic signaling. Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K+ current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K+ channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K+ channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators.

Calcium- and voltage-gated potassium (BK) channel activators in the 5β-cholanic acid-3α-ol analogue series with modifications in the lateral chain.

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate various physiological processes and represent an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel-forming subunit and thus cannot selectively target a particular tissue. We performed a structure-activity relationship study of lithocholic acid (LCA), a cholane that activates BK channels via the accessory BK β1 subunit. The latter protein is highly abundant in smooth muscle but scarce in most other tissues. Modifications to the LCA lateral chain length and functional group yielded two novel smooth muscle BK channel activators in which the substituent at C24 has a small volume and a net negative charge. Our data provide detailed structural information that will be useful to advance a pharmacophore in search of β1 subunit-selective BK channel activators. These compounds are expected to evoke smooth muscle relaxation, which would be beneficial in the pharmacotherapy of prevalent human disorders associated with increased smooth muscle contraction, such as systemic hypertension, cerebral or coronary vasospasm, bronchial asthma, bladder hyperactivity, and erectile dysfunction.

Aerobic exercise increases BKCa channel contribution to regulation of mesenteric arterial tone by upregulating β1‐subunit

Substantial evidence has shown that exercise training produces numerous adaptations within the arteries. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are expressed broadly on smooth muscle cells and play an important role in the regulation of vascular tone. The present study was designed to investigate the mechanisms by which the BK(Ca) channel underlies the exercise-induced improvement of the vascular function in mesenteric arteries. Male Wistar rats (180-200 g) were trained for 12 weeks on a treadmill (EX group) and compared with age-matched sedentary (SED) animals. Blood pressure and vascular contractility of mesenteric arteries were measured. Inside-out patch-clamp recording was applied on the freshly separated mesenteric smooth muscle cells to investigate the gating characteristics of BK(Ca) channels. Western immunoblotting was performed to study expression levels of BK(Ca) channel proteins. The maximal vascular contraction induced by noradrenaline (3 × 10(-5) M) was decreased after exercise training. Both TEA (3 × 10(-3) M) and iberiotoxin (3 × 10(-8) M) induced a significant increase of vessel tension in both SED and EX groups, but these effects were more pronounced in EX rats compared with SED animals. Inside-out patch-clamp recording showed that exercise training significantly increased the open probability, decreased the mean closed time and increased the mean open time and the sensitivity to Ca(2+) and voltage without altering the unitary conductance. The expression of BK(Ca) β1- but not α-subunit was significantly enhanced after exercise training. These data suggest that exercise training increases the contribution of the BK(Ca) channel to the regulation of vascular tone in mesenteric arteries, which is possibly mediated by upregulating β1-subunit protein to increase BK(Ca) channel activity.

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.

Smooth Muscle Cholesterol Enables BK β1 Subunit-Mediated Channel Inhibition and Subsequent Vasoconstriction Evoked by Alcohol

Hypercholesterolemia and alcohol drinking constitute independent risk factors for cerebrovascular disease. Alcohol constricts cerebral arteries in several species, including humans. This action results from inhibition of voltage- and calcium-gated potassium channels (BK) in vascular smooth muscle cells (VSMC). BK activity is also modulated by membrane cholesterol. We investigated whether VSMC cholesterol regulates ethanol actions on BK and cerebral arteries. After myogenic tone development, cholesterol depletion of rat, resistance-size cerebral arteries ablated ethanol-induced constriction, a result that was identical in intact and endothelium-free vessels. Cholesterol depletion reduced ethanol inhibition of BK whether the channel was studied in VSMC or after rat cerebral artery myocyte subunit (cbv1+β1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from β1 knockout mice were both resistant to ethanol-induced inhibition. Moreover, arteries from β1 knockout mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+β1 in bilayers and wt mouse VSMC BK were drastically blunted by cholesterol depletion. Consistently, cholesterol depletion suppressed ethanol constriction of wt mouse arteries. VSMC cholesterol and BK β1 are both required for ethanol inhibition of BK and the resulting cerebral artery constriction, with health-related implications for manipulating cholesterol levels in alcohol-induced cerebrovascular disease.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

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).

Effects of bladder outlet obstruction on properties of Ca2+-activated K+channels in rat bladder

In this study, we investigated the effects of bladder outlet obstruction (BOO) on the expression and function of large conductance (BK) and small conductance (SK) Ca(2+)-activated K(+) channels in detrusor smooth muscle. The bladder from adult female Sprague-Dawley rats with 6-wk BOO were used. The mRNA expression of the BK channel alpha-subunit, beta1-, beta2-, and beta4-subunits and SK1, SK2, and SK3 channels were investigated using real-time RT-PCR. All subunits except for the BK-beta2, SK2, and SK3 channels were predominantly expressed in the detrusor smooth muscle rather than in the mucosa. The mRNA expression of the BK channel alpha-subunit was not significantly changed in obstructed bladders. However, the expression of the BK channel beta1-subunit and the SK3 channel was remarkably increased in obstructed bladders. On the other hand, the expression of the BK channel beta4-subunit was decreased as the severity of BOO-induced bladder overactivity progressed. In detrusor smooth muscle strips from obstructed bladders, blockade of BK channels by iberiotoxin (IbTx) or charybdotoxin (CTx) and blockade of SK channels by apamin increased the amplitude of spontaneous contractions. These blockers also increased the contractility and affinity of these strips for carbachol during cumulative applications. The facilitatory effects elicited by these K(+) channel blockers were larger in the strips from obstructed bladders compared with control bladders. These results suggest that long-term exposure to BOO for 6 wk enhances the function of both BK and SK types of Ca(2+)-activated K(+) channels in the detrusor smooth muscle to induce an inhibition of bladder contractility, which might be a compensatory mechanism to reduce BOO-induced bladder overactivity.

Channel β2–4 subunits fail to substitute for β1 in sensitizing BK channels to lithocholate

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK α ( slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary β subunits (encoded by KCNMB1–4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate β subunit-containing BK channels, yet these steroids appear to be sensed by different types of β subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that β1 sensitizes the BK channel to μM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits (“cbv1”) with β1, β2, β3 or β4 in Xenopus laevis oocytes we now demonstrate that BK β2, β3 and β4 subunits fail to substitute for β1 in providing LC-sensitivity (150 μM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK β1 is highly expressed, while failing to do so in skeletal ( flexor digitorum brevis) muscle, where BK β1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by β1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional β1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

The BK channel accessory β1 subunit determines alcohol-induced cerebrovascular constriction

Ethanol-induced inhibition of myocyte large conductance, calcium- and voltage-gated potassium (BK) current causes cerebrovascular constriction, yet the molecular targets mediating EtOH action remain unknown. Using BK channel-forming (cbv1) subunits from cerebral artery myocytes, we demonstrate that EtOH potentiates and inhibits current at Cai2+ lower and higher than ∼15μM, respectively. By increasing cbv1’s apparent Cai2+-sensitivity, accessory BK β1 subunits shift the activation-to-inhibition crossover of EtOH action to <3μM Cai2+, with consequent inhibition of current under conditions found during myocyte contraction. Knocking-down KCNMB1 suppresses EtOH-reduction of arterial myocyte BK current and vessel diameter. Therefore, BK β1 is the molecular effector of alcohol-induced BK current inhibition and cerebrovascular constriction.

Promoter cloning and characterization of the rabbit BK channel β1 subunit gene

The β1 subunit of the voltage-dependent and Ca 2+-activated large-conductance K + channel (BK) in mammalian smooth muscle cells (SMCs) plays an important role in regulating smooth muscle tone and is closely linked with a series of smooth muscle tone associated diseases. However, knowledge of the transcriptional regulation of the BK β1 is still largely unclear. For the first time, we cloned and characterized the full-length genomic sequence of the rabbit BK β1 containing a 5′-flanking region of 2021 bp. The full-reading frame of the BK β1 spans ~ 7.7 kb and is organized into 4 exons and 3 introns. All of the exon/intron junction sequences contain the GT/AG consensus junction sequence. The transcription initiation site (+ 1G) is located at 447 bp upstream of the translation initiation codon. Bioinformatics analysis indicated that, without any canonical TATA-box, the 5′-flanking region possesses a high GC content and contains a number of putative transcription factor binding sites. 5′-deletion analysis demonstrated that the region of − 93/+ 30 potentially functions as a core promoter region. A gel mobility shift assay and chromatin immunoprecipitation assay revealed that Sp1 specifically interacts with a putative Sp1-binding site (− 91/− 85) in vitro and in vivo. Mutation of this site significantly diminished the promoter activities. Over-expression of Sp1 in smooth muscle cells of rabbit sphincter of Oddi enhanced the promoter activities of the BK β1 in a dose-dependent manner. Thus, we suggest that the Sp1-binding site (− 91/− 85) is essential to the basal transcription of the rabbit BK β1. Our studies provide a basic knowledge of the transcription regulation of the rabbit BK β1.

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).

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.

Molecular profile of vascular ion channels after experimental subarachnoid hemorrhage.

Cerebral vasospasm is a transient, delayed constriction of cerebral arteries that occurs after subarachnoid hemorrhage (SAH). Smooth muscle cells show impaired relaxation after SAH, which may be caused by a defect in the ionic mechanisms regulating smooth muscle membrane potential and Ca(2+) permeability. We tested this hypothesis by examining changes in expression of mRNA and protein for ion channels in the basilar arteries of dogs after SAH using quantitative real-time polymerase chain reaction (PCR) and western blotting. SAH was associated with a significant reduction in basilar artery diameter to 41 +/- 8% of pre-SAH diameter (P < 0.001) after 7 days. There was significant downregulation of the voltage-gated K(+) channel K(v) 2.2 (65% reduction in mRNA, P < 0.001; 49% reduction in protein, P < 0.05) and the beta1 subunit of the large-conductance, Ca(2+) - activated K(+) (BK) channel (53% reduction in mRNA, P < 0.02). There was no change in BK beta1 subunit protein. Changes in mRNA levels of K(v) 2.2 and the BK-beta1 subunit correlated with the degree of vasospasm (r(2) = 0.490 and 0.529 respectively, P < 0.05). The inwardly rectifying K(+) (K(ir)) channel K(ir) 2.1 was upregulated (234% increase in mRNA, P < 0.001; 350% increase in protein, P < 0.001). There was no significant change in mRNA expression of L- type Ca(2+) channels and the BK-alpha subunit. These data suggest that K(+) channel dysfunction may contribute to the pathogenesis of cerebral vasospasm.