The use of optical biosensors to detect modulation of Slack potassium channels by G-protein coupled receptors

Channels interact with MAP kinase and CaMKinase II pathways

Expression of the potassium channel Kv10.1, named EAG (for ether-a go-go), occurs primarily in neuronal synaptic endings [12, 13]. These channels have been shown to use their voltage-sensor domain to alter the activity of the MAP kinase signaling pathway [14]. Transfection of the eag gene into NIH 3T3 fibroblasts or C2C12 myoblasts has been shown to increase cell density and stimulate DNA synthesis [14]. This effect is mediated by an increase in p38 MAP kinase activity. Although this occurs regardless of the channels ability to conduct ions, mutations that shift the voltage-dependence of activation towards negative potentials, biasing the channel into its open state, block the proliferative effects of the channels. Thus, the EAG channel must be in a closed state in order to activate p38 MAP kinase. Channel conformation can therefore be seen as a switch for the signaling activity of EAG and a link between channel activity and intracellular signaling. The activation of the MAP kinase pathway by EAG may cause signaling molecules to associate or dissociate with the channel complex near the cell surface, thereby causing a dynamic mass redistribution. EAG channels also interact directly with the calmodulin dependent protein kinase CamKII [15].

Calmodulin is a dynamic regulator of ion channels

Calmodulin (CaM) is a calcium sensor protein involved in numerous biochemical signaling pathways. Increased calcium concentrations cause CaM to bind calcium, and the Ca²⁺-bound CaM can modulate numerous targets, including cellular signaling molecules [16]. The role of CaM in the inactivation of calcium-dependent voltage-gated calcium channels has been well described in the literature [17]. As these channels are permeable to calcium, inactivation of the channels by Ca²⁺-bound CaM provides negative feedback regulation of calcium entry into the cell by inactivating channels. CaM also functions as the calcium sensor for both small (SK) and intermediate (IK) conductance calcium-activated potassium channels [4, 5]. SK channels are gated solely by calcium, independent of membrane voltage. Because CaM is usually firmly tethered to the channel complex, changes in the activity of CaM due to changes in calcium levels would not be expected to induce binding or dissociation of the CaM protein. Nevertheless, Ca²⁺-bound CaM does activate numerous targets, including calmodulin-dependent protein kinases (CaM kinases), which may differentially interact with the tethered CaM, providing a potential link between channel activity and redistribution of mass.

Large conductance (BK) potassium channels interact with multiple signaling pathways

The large conductance calcium-activated potassium channel (also termed BK channel,) is regulated in a multicomponent signaling complex. These voltage gated K⁺ channels are modulated by calcium binding to an intracellular site or sites on the α-subunit that forms the pore. In mammals, at least four different β-subunits have been identified that modulate the channel. The Drosophila BK channel, although lacking the β-subunits, is modulated by numerous partners. c-Src and cAMP-dependent protein kinase (PKA) have been shown to regulate the BK channel by binding to and phosphorylating the channel [18]. Two other modulatory proteins, Slob [19] and Slip [20], also bind to the α-subunit of BK channels. Slob can bind to the signaling protein 14-3-3, and recruit the signaling protein to the channel complex. Differential phosphorylation states of Slob determine binding to 14-3-3, allowing for a dynamic regulation of the proteins constituting the BK channel multi-protein complex [21]. Such dynamic regulation of BK-associated proteins during cellular signaling could cause a significant redistribution of mass close to the plasma membrane.

Regulators of presenilins and of transcription can associate with potassium channels

Proteins in the KChIP (K Channel Interacting Proteins) family are ancillary subunits to the Kv4 family of potassium channels. They also have functions independent of the channel itself. Four proteins have been identified, termed KChIP1-4, which bind directly to the Kv4 α-subunits, and modulate the amplitude and kinetics of inactivation of the channels [22]. Before their role as potassium channel regulators was discovered, however, KChIPs were, found to function as Ca²⁺-binding proteins that regulate presenilins [23], then as Ca²⁺-dependent regulators of transcription [24]. Presenilin 1 and 2 are transmembrane protein components of a large, multisubuit asypartyl protease complex that has been named γ-secretase. This complex is required for the intramembranous cleavage of a number of membrane proteins [25]. These two roles of KChIPs provide a possible mechanism to link Kv4 channel activity with changes in transcription and of presenilin-dependent cleavage of membrane proteins, the latter of which is likely to be associated with a significant redistribution of mass at the plasma membrane.

G proteins bind to ion channels

G protein-coupled receptors (GPCRs) are a superfamily of cell membrane proteins that are involved in a vast number of cell signaling mechanisms. Agonist binding to the receptor causes the coupled heterotrimeric protein to dissociate, releasing the α- and βγ-subunits. Release of these subunits activates numerous downstream targets, allowing for amplification of the signal at the biochemical level. One family of potassium channels, the G protein-gated inwardly rectifying potassium channels (GIRKs), has been shown to be regulated directly by G proteins. Binding of the βγ heterodimer to the channel triggers channel opening, potassium ion flux, and hyperpolarization of the cell [26]. Another family of ion channels that are regulated by G proteins is that of the voltage-gated calcium channels (VGCCs), which like the GIRKS, are regulated by direct binding of the βγ heterodimer to the channel [27].

Activation of GPCRs is expected to result in a redistribution of mass at the membrane as the α- and βγ-subunits either dissociate or associate with the receptor. Previous research has shown that activation of all three classes of G-protein coupled receptors, Gs-, Gq-, and Gi-coupled receptors, cause a DMR signal that is readily detected with the optical biosensor technique [8].

Properties of the sodium-activated potassium (K_Na) channels Slack and Slick

We have given a brief and selective account of channel-protein interactions that could provide dynamic changes in mass distribution following activation of signaling pathways. More comprehensive reviews of this subject are to be found in Kaczmarek (2006) [6] and Levitan (2006) [2]. We shall now describe the properties of a class of channels that have very large cytoplasmic domains, which are potently regulated by GPCRs, and for which changes in DMR using optical biosensors can readily be detected following activation of these receptors and following direct activation of the channels themselves. These are the sodium-activated potassium channels encoded by the Slack and Slick genes (Fig. 1).

Although the existence of sodium-activated potassium currents has been known for over 20 years [28], the molecular basis of these channels has been identified only relatively recently. Our laboratory has identified the genes for two sodium-activated potassium (K_Na) channels, Slack and Slick, that are highly expressed in the central nervous system [29]. These potassium channels are sometimes described as members of the Slo family of potassium channels, and within the literature, have also been referred to as Slo2.2 (Slack) and Slo2.1 (Slick). Slack (Sequence like a Ca⁺-activated K⁺-channel) and Slick (Sequence like an intermediate conductance K⁺ channel) share only a 7% sequence homology with their nearest homologue, the large conductance (BK) channel (Slo) [30–32]. Slack and Slick do not possess the positively charged S4 domain characteristic of other voltage-sensitive channels. Unlike Slo, the N-termini of Slack and Slick are believed to reside in the cytosol. Slack is the largest K⁺ channel subunit known, and contains two regulators of K⁺ conductance (RCK) domains in its large C terminal domain [33]. Slack also has a consensus PDZ-binding motif at the end of its C-terminal tail, which may be functionally important, since Na⁺ channels and glutamate receptors, which conduct the K_Na activator Na⁺, are also known to cluster by binding to PDZ domains [34].

Slack cDNA has been expressed in mammalian HEK and CHO cells and Xenopus oocytes, and the whole cell current of these transfected cells indicates an instantaneous component followed by a slow, time dependent increase in current [30–32]. Inside-out patches of Slack-B channels have a conductance of ~180 pS in symmetrical potassium solution, and sub-conductance states that are detected frequently and range from 30–60 pS. Although Slack does not possess a positively charged S4 domain, it is outwardly-rectifying in both physiological and in symmetrical saline solutions. Cytoplasmic addition of Na⁺ results in a dose dependent increase of Slack-B activity that is reversible upon lowering Na⁺ concentration. Although channel activity is enhanced, unitary conductance remains unchanged, as would be expected for K⁺ selective channels. Slack-B channel activity cannot be detected in the absence of cytoplasmic Na⁺ [32]. Concentration-response curves for activation of Slack-B give an effective concentration of Na⁺ for 50% activation (EC₅₀) of ~41mM, and Slack-B binds Na⁺ cooperatively, with a Hill coefficient of 2.4. Both values are within the range of K_Na channels found in native tissues [29].

There are multiple splice isoforms of the Slack channel. The Slack gene is encoded by 30 exons, and in humans is located on chromosome 9q34.3. The two that have been most intensely investigated are Slack-A and Slack-B [35]. The exon encoding the N-terminal of Slack-B is located ~10 kb away from the next exon. Through searching gene databases with the N-terminal of Slick, we found another putative Slack exon 15 kb upstream of the N-terminal of Slack-B. The coding region of this exon is almost identical (>95%) to the N-terminus of Slick. This isoform of Slack was designated Slack-A, and encodes a protein of 1,203 amino acids with the N-terminal nearly identical to that of Slick, and the remainder of the sequence identical to Slack-B. In contrast to Slack-B, current in Slack-A transfected cells activate very rapidly with step changes in voltages, similar to that of Slick whole cell currents, and unlike Slack-B currents which have a slow component.

Native K_Na channels in the auditory system have been shown to adapt the pattern of neuronal firing to different rates of stimulation and influence the fidelity of information transmitted [36]. Computer modeling and biophysical and pharmacological experiments has also shown the Slack and Slick channels regulate the accuracy of firing of these neurons. In addition to these functions, the very large cytoplasmic domain of Slack has been shown to interact with a variety if cytoplasmic signaling molecules, making the channel an attractive candidate for determining if activation or modulation of these ion channels can influence the DMR in optical biosensor assays.

Slack and Slick are potently regulated by GPCRs

The activity of both Slack and Slick channels has been shown to be very strongly influenced by activation of Gαq protein coupled receptors such as the M1 muscarinic receptor and the mGluR1 metabotropic glutamate receptor, as well as by direct activation of protein kinase C (PKC) [11]. Immunocytochemical studies of brain sections and of cultured neurons have demonstrated co-expression and co-localization of these channel subunits with these GPCRs. Experiments in which the channels were co-expressed with these GPCRs in the Xenopus oocyte expression system demonstrated that Slick and Slack channels are modulated in opposite ways. Activation of these receptors by PKC produces a strong reduction of Slick currents. In contrast, activation of the same pathways produces a 2–3 fold increase in the amplitude of Slack currents and significantly slows the rate at which these current activate on depolarization. By construction of chimeric channels containing the N-terminal and membrane spanning domains of Slack with the C-terminal cytoplasmic domain of Slick, it was demonstrated that this differential modulation by GPCRs and PKC reside in the C-terminal domain and the specific phosphorylation sites responsible for this modulation are being characterized [37].

Expression of Slack alters the DMR response that follows GPCR activation

Previous research has shown that upon activation of all three classes of G-protein coupled receptors, Gs-, Gq-, and Gi-coupled receptors, can be detected with the optical biosensor technique (Epic system). To determine whether the DMR produced by activation of GPCRs can be altered by the presence of ion channels that are modulated by receptor activation we have compared the pattern and magnitude of the DMR induced by the mucarinic agonist carbamylcholine chloride (carbachol) in control HEK-293 cells with that in HEK-293 cells expressing the Slack-B potassium channel.

In order to verify that Slack channel subunits were expressed at the plasma membrane in the HEK-293 cells stably expressing the Slack-B gene, we first used conventional patch-clamping methods to verify that typical sodium-activated Slack potassium currents could be recorded from these cells. These Slack-B expressing HEK cells do not differ in apparent morphological or growth properties from the non-transfected (control) HEK cells. Both the Slack-B expressing HEK cells and the control HEK cells were plated onto a Corning Epic plate at equal densities and allowed to incubate overnight in order to generate a single monolayer of cells covering the biosensor. Then cells were incubated at 26° C in the Epic system for 2 hours prior to assay measurements. Baseline optical measurements were taken for 4.5 minutes, after which the plate was removed from the measuring system and compounds were rapidly added with an automated liquid handling system. The plate was returned to the machine and measurements were taken for the next 90 minutes.

Figure 2A demonstrates that simply removing either the control or the Slack-expressing cells from the measurement system, washing them with external medium, and then replacing them for further measurements produced no significant change in the DMR measurement. Application of carbachol to control HEK cells resulted in a positive increase in DMR, that has been described previously [38] (Fig 2B). This control response peaked at ~10 minutes and then decayed slowly over the remainder of the 90 minute measuring period (calculated t_0.5 = 35.6 min). In the Slack-B expressing cells, application of carbachol also produced a positive shift in the DMR (Fig 2C). Compared to the control cells, however, the response peaked earlier (at ~5 minutes) and was of a greater magnitude (p<0.001). Decay from the peak response in the Slack-B transfected cells also differed from that in control cells. Rather than a single slow decay, the DMR signal in Slack-B transfected cells first decayed rapidly for ~ 15 minutes from its peak (calculated t_0.5 = 17.8 min), and then decayed slowly at a rate comparable to that in control for the remainder of the 90 minute measuring period (calculated t_0.5 = 41.4 min).

We also compared the concentration-response relations for the action of carbachol on the DMR in control and Slack-expressing HEK cells (Fig. 3A,B). The differences in the kinetics of the response, with the existence of an early rapid peak in the Slack-expressing cells were apparent at all concentrations of carbachol.

We also carried out similar experiments using another agonist of GPCRs in HEK cells. SFLLR, an N-terminal TRAP pentapeptide which exhibits agonist activity against the GPCR endogenous protease activated receptor 1 (PAR1), was also applied at a concentration of 5 μM. In control untransfected HEK cells, the time course of the DMR response to SFLLR differed from that of carbachol in that the response was more sustained without a significant slow decay over measurement lasting up to 90 minutes. As with carbachol, however, expression of Slack-B altered the kinetics and amplitude of DMR upon activation of this GPCR (Fig. 4). A significant increase in the DMR occurred in Slack-B transfected cells, as compared with control cells (p<0.0001).

In summary, the presence of the Slack-B channel significantly alters the DMR that occurs upon activation of endogenous GPCRs in HEK cells. With Slack-B expressing cells the observed DMR is larger in amplitude and differs in its kinetic behavior from that observed in untransfected cells.

Reagents

Carbamoylcholine chloride (Carbachol) was purchased from Sigma Chemical Inc., St. Louis, MO. SFLLR peptide was purchased from Ana Spec, San Jose, CA. Corning Epic 384 well biosensor microplates, coated with Fibronectin, were purchased from Corning, Inc., Corning, NY.

Cell Culture

Human embryonic kidney 293 (HEK-293) cells were purchased from American Type Cell Culture. HEK-293 cells stably expressing rat Slack-B were generously donated by Yangyang Yan in the laboratory of Dr. Fred Sigworth at Yale University. For cell culturing, cells were grown in one half Dulbecco’s Modified Eagle medium (Gibco #11965) and one half Leibovitz’s L-15 Medium (Gibco #11415) supplemented with 10% heat inactived fetal bovie serum and antibiotics. 300 μg/mL Geneticin (Gibco #11811-031) was added to HEK-293 cells stably expressing Slack-B to select for expression of the Slack channel. Cells were plated on the Corning Epic biosensor plates at 12,000 cells per well and incubated overnight at 37° C under air/5% CO₂ to provide a single monolayer of cells covering the biosensor. Cells were washed 5x with Hanks Balanced Salt Solution, with calcium chloride and magnesium chloride (HBSS, Gibco #14025), with a Cy-Bi® Well 384 Well automated liquid handling system. Before assay measurements, cells plated to the Corning Epic biosensor plates were incubated in the Epic system for 2 hours at 26° C.

Dynamic Mass Redistribution (DMR) Optical Biosensor Assays

The Epic plates were removed from incubation and cells were washed 5x with 1x HBSS by a Cy-Bi Well 384 Well automated liquid handling system and allowed to incubate at 26° C in the Epic system for 2 hours prior to assay measurements. Details of the instrumental setup and assay protocols have been previously described (Fang, Ferrie, Fontaine, & Yuen, 2005.) All compound solutions were prepared in HBSS and added to cells with a Cy-Bi® Well 384 Well automated liquid handling system.

To calculate the rate of change of the DMR signal in untransfected HEK cells over time (Figures 2 and 3), we calculated a half time of decay (t_0.5) using a single exponential fit (N(t) = N₀2^−t/t0.5) to all of the data points after the peak response to agonist had occurred. For Slack-B transfected cells, in which two decay rates could clearly be differentiated, single exponential fits, using the same equation were calculated separately for the first 15 min following the peak agonist response, and for the decay that occurred subsequent to this 15 min period.