hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

A human intermediate conductance calcium-activated potassium channel

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid. In vivo in cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by Gβγ subunits, but instead shows constitutive activation, and (ii) is no longer a K+-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na+-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 μm. An almost identical high Cs+ sensitivity (IC50 = 90 μm) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.