Stanley G. Rane

The fibroblast intermediate conductance K(Ca) channel, FIK, as a prototype for the cell growth regulatory function of the IK channel family.

Abstract. The fibroblast intermediate conductance, calcium-activated potassium channel (FIK) is proposed here as a functional prototype for other IK channels which to date have undefined physiologic actions. FIK pharmacology in the 10T1/2-MRF4 myogenic fibroblast cell line was determined: to define the relationship of FIK to other IKs; to confirm a physiologic role for FIK; and, thus develop a hypothesis about IK channel family function. Whole cell patch-clamp electrophysiology was used to determine K0.5 values for FIK block by the structurally related peptides charybdotoxin (ChTX) (7 nm) and iberiotoxin (IbTX) (536 nm), and a new unrelated FIK inhibitor, Stichodactyla toxin (StK) (85 nm). Peptide pharmacology for FIK was consistent with that of recently cloned IKs. ChTX and StK inhibited bFGF stimulated 10T1/2-MRF4 cell proliferation with dose-dependencies consistent with their FIK blocking actions. ChTX, StK, and IbTX also evoked MRF4-dependent transcription as measured by muscle acetylcholine receptor channel functional expression; but they did not evoke subsequent multinucleated fiber formation or myosin heavy chain expression, suggesting a role for FIK in early, rather than late, myogenic events. Thus despite structural differences, ChTX, IbTX, and StK have common effects on cell growth and differentiation reflecting their common FIK blocking action. We suggest that a major function of the IK channel family is to regulate cell growth.

Opioid peptide modulation of Ca2+-dependent K+ and voltage-activated Ca2+ currents in bovine adrenal chromaffin cells

Opioid peptides are abundantly expressed in the adrenal medulla, and there is evidence that they may be released presynaptically or as medullary paracrine agents. To assess the physiological relevance of these observations, we investigated opioid effects on ionic currents from cultured bovine adrenal chromaffin cells. Under whole-cell path-clamp conditions, opioid peptides, acting via a mu-type opioid receptor, strongly potentiated the large conductance Ca(2+)-dependent K+ (BK) channel current. Opioids also inhibited voltage-activated Ca2+ currents. Application of opioid peptides to the extracellular face of outside-out patches also increased opening activity of single BK channels, suggestive of tight receptor-channel coupling. This potentiating effect on BK current, combined with the inhibition of Ca2+ current, indicates that opioids may have an inhibitory influence on secretory activity of the adrenal medulla. The widespread distribution of the BK channel class suggests that the significance of its modulation by opioids could also extend beyond the adrenal gland.

Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor

BACKGROUND The P4 strain of the corn smut fungus, Ustilago maydis, secretes a fungal toxin, KP4, encoded by a fungal virus (UMV4) that persistently infects its cells. UMV4, unlike most other (non-fungal) viruses, does not spread to uninfected cells by release into the extracellular milieu during its normal life cycle and is thus dependent upon host survival for replication. In symbiosis with the host fungus, UMV4 encodes KP4 to kill other competitive strains of U. maydis, thereby promoting both host and virus survival. KP4 belongs to a family of fungal toxins and determining its structure should lead to a better understanding of the function and evolutionary origins of these toxins. Elucidation of the mechanism of toxin action could lead to new anti-fungal agents against human pathogens. RESULTS We have determined the atomic structure of KP4 to 1.9 A resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel alpha-helices lying at approximately 45 degrees to these strands. The structure has two left-handed beta alpha beta cross-overs and a basic protuberance extending from the beta-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2+ and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. CONCLUSIONS Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2+ channel inhibitors may be useful lead compounds for new anti-fungal agents.

EGF IN COMBINATION WITH DEPOLARIZATION OR CAMP PRODUCES MORPHOLOGICAL BUT NOT PHYSIOLOGICAL DIFFERENTIATION IN PC12 CELLS

In response to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) receptor activated Ras/extracellular signal‐regulated kinase (ERK) signaling, PC12 cells undergo a prototypical neuronal differentiation program, characterized by neurite extension and upregulation of voltage‐gated ion channels. The epidermal growth factor (EGF) receptor also activates Ras/ERK signaling, but produces proliferation instead of differentiation. In the presence of depolarizing concentrations of KCI, however, EGF elicits neurite outgrowth through the synergistic actions of the Ras/ERK and cAMP signaling pathways. To assess if EGF and KCI/cAMP elicit the same suite of differentiation events as does NGF and bFGF, we used patch clamp recording to determine if EGF in the presence of KCI or a cAMP agonist also induced physiological differentiation as defined by upregulation of ion channels. Chronic NGF treatment of PC12 cell cultures elicited robust morphological differentiation, a threefold increase in mean calcium channel current density, and an eightfold increase in mean sodium channel current density. Sibling cultures chronically treated with EGF in the presence of high KCI or a cAMP agonist also displayed morphological differentiation, but had calcium channel current densities which were no larger than untreated, undifferentiated cells. Additionally, the increase in mean sodium channel current density induced by EGF in the presence of KCI or cAMP was no greater than the increase observed with EGF alone. Thus, although EGF in the presence of KCI or cAMP is sufficient to induce morphological differentiation as defined by neurite outgrowth, synergism of the Ras/ERK and cAMP/PKA signaling pathways is not sufficient to promote the fully physiologically differentiated PC12 phenotype. J. Neurosci. Res. 47:16–26, 1997.

Ca2+-dependent K+ Channels in Bovine Adrenal Chromaffin Cells are Modulated by Lipoxygenase Metabolites of Arachidonic Acid

Abstract. Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to potentiate activity of the Ca2+ and voltage-dependent, large-conductance K+ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca2+ buffering ruled out cytoplasmic messengers or changes in intracellular Ca2+ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting action potential duration and thus voltage-gated Ca2+ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs.

Cyanide interaction with redox modulatory sites enhances NMDA receptor responses.

Activation of NMDA receptors plays an important role in cyanide neurotoxicity. Cyanide indirectly activates the receptor by inducing neuronal release of glutamate and also enhances receptor‐mediated responses by a direct interaction with the receptor complex. This study investigated the mechanism in cerebellar granule cells by which cyanide enhances NMDA‐induced Ca2+ influx. Cyanide (50 μM) increased the influx of Ca2+ over the NMDA concentration range of 0.5–500 μM. Experiments showed that cyanide does not interact with the receptors glycine or PKC mediated phosphorylation regulatory sites. N‐ethylmaleimide, a thiol alkylating agent which inactivates the redox regulatory sites of the receptor, blocked the enhancing effect of cyanide. Pretreatment of cells with 5,5‐dithio‐bis‐2‐nitrobenzoic acid (DTNB), a compound that oxidizes the receptor redox sites, had no effect on the response to cyanide. On the other hand, the nonpermeant reducing agents, dithiothreitol or cysteine, further increased the cyanide effect. These observations can be explained by cyanide interacting with redox sensitive disulfide groups that are not accessible to the non‐permeant reducing agents. It is proposed that cyanide interacts with a redox site(s) located either on the intracellular receptor domain or in the transmembrane hydrophobic domain. Furthermore the enhancement by cyanide of the excitotoxic actions of NMDA involves receptor sites that are sensitive to oxidation/reduction and this interaction contributes to the neurotoxic action of cyanide.

The Small Conductance Calcium-activated Potassium Channel Regulates Ion Channel Expression in C3H10T1/2 Cells Ectopically Expressing the Muscle Regulatory Factor MRF4

We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factor-induced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.

Potassium channels as targets for modulating cell growth and differentiation

Potassium channels, especially small and intermediate conductance KCa channels, have important roles in controlling cell proliferation and differentiation. Thus far regulation of these channels is reported to be primarily at the level of expression, in response to activation of the central growth regulatory signaling pathway (i.e., growth factor receptor tyrosine kinase/Ras/Raf/MEK/ERK). Therefore, the function and regulation of these cell growth-associated channels must be viewed differently from that of channels which govern electrical signaling in excitable cells, and which are typically studied in terms of their transient modulation by G-protein coupled receptors. Although there are suggestions that potassium channels also contribute to growth regulation in excitable cells, a coherent picture of this role in these systems is still emerging. For fibroblasts and T-lymphocytes, it is clear that growth factor and oncogenic upregulation of a unique KCa channel (or possibly KCa channel class) is stimulatory for cell proliferation and activation, respectively. This mitogenic channel has a single channel conductance in the range of 33–39 pS, it is charybdotoxin-sensitive and apamin-insensitive, and its gating is voltage-independent. Recent cloning data suggest that the KCa channel (or channel class) described for fibroblasts and T-cells has a widespread distribution in other mitogenically active (non-neuronal) tissues. A number of studies are now underway to understand the physiology, pharmacology and regulation of this channel. Further, it is now critical to determine how KCa activity integrates into the signaling pathways which convey growth regulatory information from the cell membrane, to the nucleus, and then to the ultimate effectors for cell proliferation or differentiation. It has also become apparent that these growth regulatory signaling systems interact with other channel types, affecting channel densities at the level of expression, and channel activities at the level of gating modulation. Therefore, it seems most appropriate to view ion channel function in the context of how it contributes to and is affected by both cell growth activity, and the biochemical signaling systems linked to growth control.