Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.     

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.     

Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L- type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.     

Molecular architecture and divalent cation activation of TvoK, a prokaryotic potassium channel

RCK (regulator of conductance of potassium) domains form a family of ligand-binding domains found in many prokaryotic K+ channels and transport proteins. Although many RCK domains contain an apparent nucleotide binding motif, some are known instead to bind Ca2+, which can then facilitate channel opening. Here we report on the molecular architecture and ligand activation properties of an RCK-containing potassium channel cloned from the prokaryote Thermoplasma volcanium. This channel, called TvoK, is of an apparent molecular mass and subunit composition that is consistent with the hetero-octameric configuration hypothesized for the related MthK (Methanobacterium thermoautotrophicum potassium) channel, in which four channel-tethered RCK domains coassemble with four soluble (untethered) RCK domains. The expression of soluble TvoK RCK subunits arises from an unconventional UUG start codon within the TvoK gene; silent mutagenesis of this alternative start codon abolishes expression of the soluble form of the TvoK RCK domain. Using single channel recording of purified, reconstituted TvoK, we found that the channel is activated by Ca2+ as well as Mg2+, Mn2+, and Ni2+. This non-selective divalent activation is in contrast with the activation properties of MthK, which is selectively activated by Ca2+. Transplantation of the TvoK RCK domain into MthK generates a channel that can be activated by Mg2+, illustrating that the Mg2+ binding site is likely contained within the RCK domain. We present a working hypothesis for TvoK gating in which the binding of either Ca2+ or Mg2+ can contribute approximately 5 kcal/mol toward stabilization of the open conformation of the channel.     

Characterization of a Shaw-related potassium channel family in rat brain.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.     

Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C.     

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Actions of putative chloride channel blocking agents on canine lower esophageal sphincter (LES)

Niflumic acid (NA), a putative Cl(-)-channel blocker, has provided pharmacological evidence that Cl(-)-channel closures mediate hyperpolarization caused by NO in gastrointestinal smooth muscle. However, NA caused concentration-dependent relaxation of canine lower esophageal sphincter (LES) and failed to inhibit NO-mediated relaxations. DIDS also did not inhibit NO-mediated relaxations, but did abolish them when present with 20 mM TEA (tetraethyl ammonium ion), which was also ineffective alone. TEA reversed NA-induced relaxations, but with NA it did not inhibit NO-mediated relaxations. We investigated the modes of action of these agents further. Neither nerve-function block nor block of NOS activity affected the inhibition of LES tone by NA. In patch-clamp studies, NA increased outward currents from -30 to + 90 mV when [Ca2+]pipette was 50 nM. This was prevented by 20 mM TEA, but not by prior inhibition of NOS. At 200 nM [Ca2+]pipette, TEA markedly reduced outward currents, but did not prevent the increase from subsequent NA. In contrast, under similar conditions, application of DIDS after 20 mM TEA further reduced outward currents. When the patch pipette contained CsCl and TEA to block K+ currents, NA had no significant effect on currents between -50 and +90 mV. Thus, NA acted by opening K+ channels: some TEA-sensitive and some not. It had no detectable effect on currents when K+ channels were blocked. We conclude that NA is an unreliable pharmacological tool to evaluate Cl(-)-channel contributions to smooth muscle function. DIDS did not open K+ channels. Decreases in outward currents from DIDS may result from inhibition of K+ currents or currents carried by Cl- at depolarized membrane potentials.     

Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.     

A new class of calcium entry blockers defined by 1,3-diphosphonates. Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.     

Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces

In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions. Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization. The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd. MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability. Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2). MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio. Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge. These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.     

Potassium currents regulating secretion from Brunner's glands in guinea pig duodenum

This study examined the role of outward K(+) currents in the acinar cells underlying secretion from Brunner's glands in guinea pig duodenum. Intracellular recordings were made from single acinar cells in intact acini in in vitro submucosal preparations, and videomicroscopy was employed in the same preparation to correlate these measures with secretion. Mean resting membrane potential was -74 mV and was depolarized by high external K(+) (20 mM) and the K(+) channel blockers 4-aminopyridine (4-AP), quinine, and clotrimazole. The cholinergic agonist carbachol (60-2,000 nM; EC(50) = 200 nM) caused a concentration-dependent initial hyperpolarization of the membrane and an associated decrease in input resistance. This hyperpolarization was significantly decreased by 20 mM external K(+) or membrane hyperpolarization and increased by 1 mM external K(+) or membrane depolarization. It was blocked by the K(+) channel blockers tetraethylammonium (TEA), 4-AP, quinine, and clotrimazole but not iberiotoxin. When videomicroscopy was employed to measure dilation of acinar lumen in the same preparation, carbachol-evoked dilations were altered in a parallel fashion when external K(+) was altered. The dilations were also blocked by the K(+) channel blockers TEA, 4-AP, quinine, and clotrimazole but not iberiotoxin. These findings suggest that activation of outward K(+) currents is fundamental to the initiation of secretion from these glands, consistent with the model of K(+) efflux from the basolateral membrane providing the driving force for secretion. The pharmacological profile suggests that these K(+) channels belong to the intermediate conductance group.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Structures of the MthK RCK domain and the effect of Ca2+ on gating ring stability

MthK is a Ca2+-gated K+ channel from Methanobacterium autotrophicum. The crystal structure of the MthK channel in a Ca2+-bound open state was previously determined at 3.3 A and revealed an octameric gating ring composed of eight intracellular ligand-binding RCK (regulate the conductance of K+) domains. It was suggested that Ca2+ binding regulates the gating ring conformation, which in turn leads to the opening and closing of the channel. However, at 3.3 AA resolution, the molecular details of the structure are not well defined, and many of the conclusions drawn from that structure were hypothetical. Here we have presented high resolution structures of the MthK RCK domain with and without Ca2+ bound from three different crystals. These structures revealed a dimeric architecture of the RCK domain and allowed us to visualize the Ca2+ binding and protein-protein contacts at atomic detail. The dimerization of RCK domains is also conserved in other RCK-regulated K+ channels and transporters, suggesting that the RCK dimer serves as a basic unit in the gating ring assembly. A comparison of these dimer structures confirmed that the dimer interface is indeed flexible as suggested previously. However, the conformational change at the flexible interface is of an extent smaller than the previously hypothesized gating ring movement, and a reconstruction of these dimers into octamers by applying protein-protein contacts at the fixed interface did not generate enclosed gating rings. This indicated that there is a high probability that the previously defined fixed interface may not be fixed during channel gating. In addition to the structural studies, we have also carried out biochemical analyses and have shown that near physiological pH, isolated RCK domains form a stable octamer in solution, supporting the notion that the formation of octameric gating ring is a functionally relevant event in MthK gating. Additionally, our stability studies indicated that Ca2+ binding stabilizes the RCK domains in this octameric state.     

Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel

The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 A resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ (here referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+ activated K+ channels.     

A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels

Endothelial cells from brain microvessels form the blood-brain barrier. Brain microvessels and endothelial cells isolated from rat brain microvessels express an amiloride-sensitive cationic channel that was characterized using [3H]phenamil binding and patch-clamp experiments. [3H]Phenamil, a labeled amiloride analog, recognizes a single family of binding sites with a dissociation constant of 20-30 nM and a maximum binding capacity of 8-15 pmol/mg protein. The pharmacological profile of the channel (phenamil greater than benzamil greater than amiloride) is very similar to that of the epithelium Na+ channel of mammalian kidney and of frog epithelia. Long-lasting currents were observed in patch-clamp experiments using excised outside-out patches. Application of amiloride or phenamil first produced a rapid flickering of channel activity and then its complete blockade. The mean unit channel conductance at 140 mM Na+ was 23 picosiemens. The selectivity of Na+ over K+ was estimated from reversal potentials to be 1.5:1. Properties of the channel in microvessels are clearly distinct from those of the Na+ channel of the kidney, suggesting the existence of several isoforms of cationic channels that are sensitive to amiloride and its derivatives. The low selectivity cationic channel of endothelial cells in brain microvessels might be important for controlling both Na+ and K+ movements across the blood-brain barrier.     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.