Effects of trimethoprim-sulfadiazine and detomidine on the function of equine Kv 11.1 channels in a two-electrode voltage-clamp (TEVC) oocyte model.

The long QT syndrome (LQTS) is a channelopathy that can lead to severe arrhythmia and sudden cardiac death. Pharmacologically induced LQTS is caused by interaction between drugs and potassium channels, especially the Kv 11.1 channel. Due to such interactions, numerous drugs have been withdrawn from the market or are administered with precautions in human medicine. However, some compounds, such as trimethoprim-sulfonamide combinations are still widely used in veterinarian medicine. Therefore, we investigate the effect of trimethoprim-sulfadiazine (TMS), trimethoprim, sulfadiazine, and detomidine on equine-specific Kv 11.1 channels. Kv 11.1 channels cloned from equine hearts were heterologously expressed in Xenopus laevis oocytes, and whole cell currents were measured by two-electrode voltage-clamp before and after drug application. TMS blocked equine Kv 11.1 current with an IC50 of 3.74 mm (95% CI: 2.95-4.73 mm) and affected the kinetics of activation and inactivation. Similar was found for trimethoprim but not for sulfadiazine, suggesting the effect is due to trimethoprim. Detomidine did not affect equine Kv 11.1 current. Thus, equine Kv 11.1 channels are also susceptible to pharmacological block, indicating that some drugs may have the potential to affect repolarization in horse. However, in vivo studies are needed to assess the potential risk of these drugs to induce equine LQTS.

Differences in the electrocardiographic QT interval of various breeds of athletic horses during rest and exercise.

OBJECTIVES: Quantitative measurements of cardiac repolarization, defined as the electrocardiographic QT interval, have important diagnostic implications in humans, as irregularities can trigger potentially fatal ventricular tachyarrhythmia. In both humans and horses, cardiac repolarization is influenced to some extent by heart rate, age, body weight (BW), sex, autonomic tone, and environment. In horses, there is substantial inter-breed variation in size and training, and the aims of this study were therefore to determine the best model describing the QT to RR relationship in breeds of various athletic horses and to test for differences in the QT interval. ANIMALS: Ten Icelandic horses, 10 Arabian horses, 10 Thoroughbreds, 10 Standardbreds, six Coldblood trotters, 10 Warmbloods (dressage) and 10 Warmbloods (show jumping). All horses were geldings. METHODS: QT intervals were measured from resting to peak exercise level and plotted against RR intervals. Data points were fitted with relevant regression models, and the effect of breed, BW, and estimated exercise intensity was examined. RESULTS: For all breeds in this study, the QT interval was best described as a function of RR by the piecewise linear regression model. The breed of horse had a significant effect on the model. There was no systematic effect of BW or estimated exercise intensity, but a high inter-horse variability was observed. CONCLUSIONS: The equine QT interval should preferably be corrected for heart rate according to breed. In addition, the results indicate that equine studies of the QT interval must be designed to eliminate the influence of a large inter-horse variation.

Purinergic signalling - a possible mechanism for KCNQ1 channel response to cell volume challenges.

AIM: A number of K(+) channels are regulated by small, fast changes in cell volume. The mechanisms underlying cell volume sensitivity are not known, but one possible mechanism could be purinergic signalling. Volume activated ATP release could trigger signalling pathways that subsequently lead to ion channel stimulation and cell volume back-regulation. Our aim was to investigate whether volume sensitivity of the voltage-gated K(+) channel, KCNQ1, is dependent on ATP release and regulation by purinergic signalling. METHODS: We used Xenopus oocytes heterologously expressing human KCNQ1, KCNE1, water channels (AQP1) and P2Y2 receptors. ATP release was monitored by a luciferin-luciferase assay and ion channel conductance was recorded by two-electrode voltage clamp. RESULTS: The luminescence assay showed that oocytes released ATP in response to mechanical, hypoosmotic stimuli and hyperosmotic stimuli. Basal ATP release was approx. three times higher in the KCNQ1 + AQP1 and KCNQ1 injected oocytes compared to the non-injected ones. Exogenously added ATP (0.1 mm) did not have any substantial effect on volume-induced KCNQ1 currents. Nevertheless, apyrase decreased all currents by about 50%. Suramin inhibited about 23% of the KCNQ1 volume sensitivity. Expression of P2Y2 receptors stimulated endogenous Cl(-) channels, but it also led to 68% inhibition of the KCNQ1 currents. Adenosine (0.1 mm) also inhibited the KCNQ1 currents by about 56%. CONCLUSION: Xenopus oocytes release ATP in response to mechanical stimuli and cell volume changes. Purinergic P2 and P1 receptors confer some of the KCNQ1 channel volume sensitivity, although endogenous adenosine receptors and expressed P2Y2 receptors do so in the negative direction.

Localization of large conductance calcium-activated potassium channels and their effect on calcitonin gene-related peptide release in the rat trigemino-neuronal pathway.

Large conductance calcium-activated potassium (BK(Ca)) channels are membrane proteins contributing to electrical propagation through neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide found in the trigeminovascular system (TGVS). Both BK(Ca) channels and CGRP are involved in migraine pathophysiology. Here we study the expression and localization of BK(Ca) channels and CGRP in the rat trigeminal ganglion (TG) and the trigeminal nucleus caudalis (TNC) as these structures are involved in migraine pain. Also the effect of the BK(Ca) channel blocker iberiotoxin and the BK(Ca) channel opener NS11021 on CGRP release from isolated TG and TNC was investigated. By RT-PCR, BK(Ca) channel mRNA was detected in the TG and the TNC. A significant difference in BK(Ca) channel mRNA transcript levels were found using qPCR between the TNC as compared to the TG. The BK(Ca) channel protein was more expressed in the TNC as compared to the TG shown by western blotting. Immunohistochemistry identified BK(Ca) channels in the nerve cell bodies of the TG and the TNC. The beta2- and beta4-subunit proteins were found in the TG and the TNC. They were both more expressed in the TNC as compared to TG shown by western blotting. In isolated TNC, the BK(Ca) channel blocker iberiotoxin induced a concentration-dependent release of CGRP that was attenuated by the BK(Ca) channel opener NS11021. No effect on basal CGRP release was found by NS11021 in isolated TG or TNC or by iberiotoxin in TG. In conclusion, we found both BK(Ca) channel mRNA and protein expression in the TG and the TNC. The BK(Ca) channel protein and the modulatory beta2- and beta4-subunt proteins were more expressed in the TNC than in the TG. Iberiotoxin induced an increase in CGRP release from the TNC that was attenuated by NS11021. Thus, BK(Ca) channels might have a role in trigeminovascular pain transmission.

Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes.

Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels.

Pharmacological modulation of SK3 channels.

Small-conductance, calcium-activated K+ channels (SK channels) are voltage-insensitive channels that have been identified molecularly within the last few years. As SK channels play a fundamental role in most excitable cells and participate in afterhyperpolarization (AHP) and spike-frequency adaptation, pharmacological modulation of SK channels may be of significant clinical importance. Here we report the functional expression of SK3 in HEK293 and demonstrate a broad pharmacological profile for these channels. Brain slice studies commonly employ 4-aminopyridine (4-AP) to block voltage-dependent K+ channels or a methyl derivative of bicuculline, a blocker of gamma-aminobutyric acid (GABA)-gated Cl- channels, in order to investigate the role of various synapses in specialized neural networks. However, in this study both 4-AP and bicuculline are shown to inhibit SK3 channels (IC50 values of 512 microM and 6 microM, respectively) at concentrations lower than those used for brain slice recordings. Riluzole, a potent neuroprotective drug with anti-ischemic, anticonvulsant and sedative effects currently used in the treatment of amyotrophic lateral sclerosis, activates SK3 channels at concentrations of 3 microM and above. Amitriptyline, a tricyclic antidepressive widely used clinically, inhibits SK3 channels with an IC50 of 39.1 +/- 10 microM (n=6).

Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit.

1. In order to study its role in steady state water transport, the Na+-glucose cotransporter (SGLT1) was expressed in Xenopus laevis oocytes; both the human and the rabbit clones were tested. The transport activity was monitored as a clamp current and the flux of water followed optically as the change in oocyte volume. 2. SGLT1 has two modes of water transport. First, it acts as a molecular water pump: for each 2 Na+ and 1 sugar molecule 264 water molecules were cotransported in the human SGLT1 (hSGLT1), 424 for the rabbit SGLT1 (rSGLT1). Second, it acts as a water channel. 3. The cotransport of water was tightly coupled to the sugar-induced clamp current. Instantaneous changes in clamp current induced by changes in clamp voltage were accompanied by instantaneous changes in the rate of water transport. 4. The cotransported solution was predicted to be hypertonic, and an osmotic gradient built up across the oocyte membrane with continued transport; this resulted in an additional osmotic influx of water. After 5-10 min a steady state was achieved in which the total influx was predicted to be isotonic with the intracellular solution. 5. With the given expression levels, the steady state water transport was divided about equally between cotransport, osmosis across the SGLT1 and osmosis across the native oocyte membrane. 6. Coexpression of AQP1 with the SGLT1 increased the water permeability more than 10-fold and steady state isotonic transport was achieved after less than 2 s of sugar activation. One-third of the water was cotransported, and the remainder was osmotically driven through the AQP1. 7. The data suggest that SGLT1 has three roles in isotonic water transport: it cotransports water directly, it supplies a passive pathway for osmotic water transport, and it generates an osmotic driving force that can be employed by other pathways, for example aquaporins.

Apamin interacts with all subtypes of cloned small-conductance Ca2+-activated K+ channels.

The purpose of the present study was to examine how apamin interacts with the three cloned subtypes of small-conductance Ca2+-activated K+ channels (hSK1, rSK2 and rSK3). Expression of the SK channel subtypes in Xenopus laevis oocytes resulted in large outward currents (0.5-5 microA) after direct injection of Ca2+. In all three cases the Ca2+-activated K+ currents could be totally inhibited by 500 nM apamin. Dose-response curves revealed a subtype-specific affinity for the apamin-induced inhibition with IC50 values of 704 pM and 196 nM (biphasic) for hSK1, 27 pM for rSK2 and 4 nM for rSK3. Consistent with these results, membranes prepared from oocytes expressing the SK channel subtypes bound 125I-labelled apamin with distinct dissociation constants (Kd values) of approx. 390 pM for hSK1, 4 pM for rSK2 and 11 pM for rSK3. These results show that apamin binds to and blocks all three subtypes of cloned SK channels, and the distinct values for IC50 and Kd suggest that apamin may be useful for determining the expression pattern of SK channel subtypes in native tissue.

Water transport by the human Na+-coupled glutamate cotransporter expressed in Xenopus oocytes.

The water transport properties of the human Na+-coupled glutamate cotransporter (EAAT1) were investigated. The protein was expressed in Xenopus laevis oocytes and electrogenic glutamate transport was recorded by two-electrode voltage clamp, while the concurrent water transport was monitored as oocyte volume changes. Water transport by EAAT1 was bimodal. Water was cotransported along with glutamate and Na+ by a mechanism within the protein. The transporter also sustained passive water transport in response to osmotic challenges. The two modes could be separated and could proceed in parallel. The cotransport modality was characterized in solutions of low Cl- concentration. Addition of glutamate promptly initiated an influx of 436 +/- 55 water molecules per unit charge, irrespective of the clamp potential. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein primarily from the downhill fluxes of Na+ to the uphill fluxes of water. Experiments using the cation-selective ionophore gramicidin showed no unstirred layer effects. Na+ currents in the ionophore did not lead to any significant initial water movements. In the absence of glutamate, EAAT1 contributed a passive water permeability (Lp) of (11.3 +/- 2.0) x 10(-6) cm s(-1) (osmol l(-1))(-1). In the presence of glutamate, Lp was about 50 % higher for both high and low Cl- concentrations. The physiological role of EAAT1 as a molecular water pump is discussed in relation to cellular volume homeostasis in the nervous system.

Transport of water and glycerol in aquaporin 3 is gated by H(+).

Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L(p), was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. The L(p) values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeability P(Gl), obtained from [(14)C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (sigma). For AQP3, water and glycerol interacted by competing for titratable site(s): sigma(Gl) was 0.15 at neutral pH but doubled at pH 6.4. The sigma values were smaller for polyols in which the -OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol(-1). We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites.

Quantification and distribution of Ca(2+)-activated maxi K(+) channels in rabbit distal colon.

The Ca(2+)-activated maxi K(+) channel is an abundant channel type in the distal colon epithelium, but nothing is known regarding the actual number and precise localization of these channels. The aim of this study has therefore been to quantify the maxi K(+) channels in colon epithelium by binding of iberiotoxin (IbTX), a selective peptidyl ligand for maxi K(+) channels. In isotope flux measurements 75% of the total K(+) channel activity in plasma membranes from distal colon epithelium is inhibited by IbTX (K(0.5) = 4.5 pM), indicating that the maxi K(+) channel is the predominant channel type in this epithelium. Consistent with the functional studies, the radiolabeled double mutant (125)I-IbTX-D19Y/Y36F binds to the colon epithelium membranes with an equilibrium dissociation constant of approximately 10 pM. The maximum receptor concentration values (in fmol/mg protein) for (125)I-IbTX-D19Y/Y36F binding to colon epithelium are 78 for surface membranes and 8 for crypt membranes, suggesting that the maxi K(+) channels are predominantly expressed in the Na(+)-absorbing surface cells, as compared with the Cl(-)-secreting crypt cells. However, aldosterone stimulation of this tissue induced by a low-Na(+) diet does not change the total number of maxi K(+) channels.

Bidirectional water fluxes and specificity for small hydrophilic molecules in aquaporins 0-5.

The dimensions of the aqueous pore in aquaporins (AQP) 0, 1, 2, 3, 4, and 5 expressed in Xenopus laevis oocytes were probed by comparing the ability of various solutes to generate osmotic flow. By improved techniques, volume flows were determined from initial rates of changes. Identical values for the osmotic water permeability (Lp) were obtained in swelling as in shrinkage experiments demonstrating, for the first time, that aquaporins are bidirectional. The reflection coefficients (sigma) of urea, glycerol, acetamide, and formamide at 23 degreesC were: AQP0: 1, 1, 0.8, 0.6; AQP1: 1, 0.8, 1, 1; AQP2: 1, 0.8, 1, 1; AQP3: 1, 0.2, 0.7, 0.4; AQP4: 1, 0.9, 1, 1; and AQP5: 1, 1, 1, 0.8. As seen there is no clear connection between solute size and permeation. At 13 degreesC the sigmas for AQP3 were 1, 0.4, 1, and 0.5; functionally, this pore narrows at lower temperatures. HgCl2 reversibly reduced the Lp of AQP3 and increased sigmaglyc to 1 and sigmaform to 0.6. We conclude that the pore of the various aquaporins are structurally different and that a simple steric model is insufficient to explain solute-pore interactions.

The human Na+-glucose cotransporter is a molecular water pump.

1. The human Na+-glucose cotransporter (hSGLT1) was expressed in Xenopus laevis oocytes. The transport activity, given by the Na+ current, was monitored as a clamp current and the concomitant flux of water followed optically as the change in oocyte volume. 2. When glucose was added to the bathing solution there was an abrupt increase in clamp current and an immediate swelling of the oocyte. The transmembrane transport of two Na+ ions and one sugar molecule was coupled, within the protein itself, to the influx of 210 water molecules. 3. This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. 4. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein from the downhill fluxes of Na+ and sugar to the uphill transport of water, indicative of secondary active transport of water. 5. Unstirred layer effects were ruled out on the basis of experiments on oocytes treated with gramicidin or other ionophores. Na+ currents maintained by ionophores did not lead to any initial water movements. 6. The finding of a molecular water pump allows for new models of cellular water transport which include coupling between ion and water fluxes at the protein level; the hSGLT1 could account for almost half the daily reuptake of water from the small intestine.

Regulation of Ca(2+)-activated K+ channels from rabbit distal colon.

Transepithelial transport in the rabbit distal colon surface cells involves the integrated function of amiloride-sensitive Na+ channels in the luminal membrane and the Na, K-pump, together with K+ channels in the basolateral membrane. Incorporation of plasma membrane vesicles from surface cells into planar lipid bilayers shows that a Ca(2+)-activated maxi K+ channel with a single channel conductance of approximately 275 pS is predominant in the basolateral membrane of this cell type. The epithelial Ca(2+)-activated maxi K+ channels are regulated by Ca2+ in the intracellular range of concentration, pH, and membrane potential. The high Ca(2+)-sensitivity of the epithelial maxi K+ channels is dependent on the channel protein being in a phosphorylated state. Dephosphorylated channels can regain their Ca(2+)-sensitivity after phosphorylation catalyzed by a cAMP dependent protein kinase. The extensive regulation of the epithelial maxi K+ channels by intracellular factors suggests that these channels may play an important role in regulation of transepithelial transport and may be one explanation for the apparent tissue to tissue variability in properties for this channel type.

Water transport by the Na+/glucose cotransporter under isotonic conditions.

Solute cotransport in the Na+/glucose cotransporter is directly coupled to significant water fluxes. The water fluxes are energized by the downhill fluxes of the other substrates by a mechanism within the protein itself. In the present paper we investigate the Na+/glucose cotransporter expressed in Xenopus oocytes. We present a method which allows short-term exposures to sugar under voltage clamp conditions. We demonstrate that water is cotransported with the solutes despite no osmotic differences between the external and intracellular solutions. There is a fixed ratio of 195:1 between the number of water molecules and the number of Na+ ions transported, equivalent to 390 water molecules per glucose molecule. Unstirred layer effects are ruled out on the basis of experiments on native oocytes incubated with the ionophores gramicidin D or nystatin.

Identification of beta2-glycoprotein I as a membrane-associated protein in kidney: purification by calmodulin affinity chromatography.

Outer renal medulla calmodulin-binding proteins from a soluble protein fraction and a plasma membrane fraction solubilized in CHAPS were retained on a calmodulin-Sepharose 4B column in the presence of Ca2+, and subsequently eluted by EGTA. The calmodulin-binding proteins constituted 2.5% of the soluble protein and 0.1% of the solubilized membrane protein. beta2-glycoprotein I was identified as a calmodulin-binding protein both by N-terminal sequencing and by immunoblotting. Quantification showed that beta2-glycoprotein I constituted the major part (approx. 35%) of the calmodulin-binding membrane proteins, but only a minor part (approx. 0.1%) of the calmodulin-binding proteins in the soluble fraction. These results show for the first time that beta2-glycoprotein I binds calmodulin and that beta2-glycoprotein I may in kidney be a membrane-associated protein. Immunohistochemical studies identified beta2-glycoprotein I in several parts of the cortex and the medulla of the kidney, including Bowman's capsula, the tubular lumen and the tubular epithelium, indicating that beta2-glycoprotein I, despite its relatively high molecular mass, is filtrated in the glomerulus and subsequently reabsorbed by the tubular epithelium. This is in agreement with beta2-glycoprotein I being a marker for renal tubular disease.

Characterization of the interaction between beta2-glycoprotein I and calmodulin, and identification of a binding sequence in beta2-glycoprotein I.

beta2-Glycoprotein I was shown to bind reversibly to calmodulin in a Ca2+-dependent manner with a 1:1 stoichiometry, a Kd of 3 x 10(-9) M and a Hill coefficient of 1.4. A sequence in beta2-glycoprotein I (Lys-Pro-Gly-Tyr-Val-Ser-Arg-Gly-Gly-Met-Arg-Lys-Phe-Ile-) limited by Cys-32 and Cys-47 is suggested to be the calmodulin-binding region. This sequence was the only one in beta2-glycoprotein I theoretically having the ability to form a basic amphiphilic alpha-helix typical of a calmodulin binding sequence. The peptide corresponding to this sequence was synthesized and found to inhibit the interaction between beta2-glycoprotein I and calmodulin with an IC50 value of 0.38 x [beta2-glycoprotein I] and to displace the beta2-glycoprotein I from the beta2-glycoprotein I/calmodulin complex with an IC50 value of 0.90 x [beta2-glycoprotein I].

Regulation of Ca2+-activated K+ channel from rabbit distal colon epithelium by phosphorylation and dephosphorylation.

The Ca2+-activated maxi K+ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na+ reabsorption from the intestinal lumen. Previous measurements of these basolateral K+ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca2+ with a K0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca2+. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K+ channels lost their high sensitivity to Ca2+, yet they could still be activated by high concentrations of Ca2+ (10 micron). Furthermore, the high sensitivity to Ca2+ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30-120 sec for dephosphorylation and of 10-280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca2+ of the maxi K+ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca2+ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca2+ sensitivities for maxi K+ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems.

Purification and characterization of epithelial Ca(2+)-activated K+ channels.

Reabsorption of NaCl in the thick ascending limb of Henle's loop in the kidney and in the surface cells in the distal colon involves the integrated function of several membrane transport systems including ion channels, the Na,K,Cl-cotransport system and the Na,K-pump. To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-activated K+ channels from the luminal membrane of the TAL cells and from the basolateral membrane of the distal colon cells have been characterized by flux studies in plasma membrane vesicle preparations and by single channel measurements in lipid bilayers. The channels are found to be activated by Ca2+ in the physiological range of concentration with a strong dependence on intracellular pH and the membrane potential. The Ca(2+)-sensitivity of the K+ channels is modulated by phosphorylation and dephosphorylation and the K+ channel protein must be in a phosphorylated state to respond to intracellular concentrations of Ca2+. As a step towards purification of the K+ channel proteins, procedures for solubilization and reconstitution of the K+ channels have been developed. The observation that the epithelial Ca(2+)-activated K+ channels bind calmodulin in the presence of Ca2+ have allowed for partial purification of the K+ channel proteins by calmodulin affinity chromatography. In the sequences for the two cloned Ca(2+)-activated K+ channels, the mSlo channel and the slowpoke channel, putative calmodulin binding regions can be identified.

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium.

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-activated K+ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca(2+)-activated K+ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca2+ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K0.5 for Ca2+ was raised from 20 nM at a potential of 0 mV to 300 nM at -40 mV. A decrease in pH at the cytoplasmic face of the K+ channel reduced the Ca2+ sensitivity dramatically. A loss of the high sensitivity to Ca2+ was also observed after incubation with MgCl2, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca2+ sensitivity between studies on K+ channels from the same tissue. High affinity inhibition (K0.5 = 10 nM) by charybdotoxin of the Ca(2+)-activated K+ channel from the extracellular face could be lifted by an outward flux of K+ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K+ channels. The high sensitivity for Ca2+ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K+ channel in the epithelial cells.