GSBS Dissertations and Theses

Title

Mechanism of Fatty Acid Modulation of Calcium-Activated Potassium Channel Activity

Approval Date

December 1997

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Physiology

Subjects

Fatty Acids; Potassium Channels; Calcium Channels; Academic Dissertations; Dissertations, UMMS

Abstract

The purpose of this work was to determine whether the previously identified fatty acid activation of large conductance Ca2+-activated K+ (BK) channels from rabbit pulmonary artery smooth muscle cells was due to the direct interaction of the fatty acid with a site on the channel protein. If this was found to be the case, this study would also attempt to identify the site of fatty acid-protein interaction.

Fatty acids released from membrane phospholipids by cellular phospholipases or available to the cell from the extracellular environment are important signaling molecules. Fatty acids can modulate the activity of a large number of molecules including protein kinases, phospholipases, adenylate and guanylate cyclases, G-proteins and ion channels. Fatty acids have also been shown to activate transcription of genes belonging to the steroid/thyroid superfamily of receptors. The actions of fatty acids on signal transduction pathways can be direct, whereby the fatty acid molecule itself is responsible for changes in the activity of enzymes, ion channels and other proteins. Alternatively, the effects of fatty acids may be indirect. In this case, biologically active lipids, produced from the metabolism of arachidonic acid are responsible for changes in cellular signaling. A previous study on the fatty acid modulation of rabbit pulmonary artery smooth muscle BK channel activity concluded that channel activation by fatty acids did not involve cycloxygenase, lip oxygenase and P450 metabolites (122), eliminating this indirect action of fatty acids as a possible mechanism.

When dealing with the effects of fatty acids on membrane bound ion channel proteins, other mechanisms of action are also possible. For example, fatty acids are capable of entering the cell membrane and can thus affect properties of the lipid bilayer, such as membrane fluidity or membrane surface charge, that may consequently alter the activity of ion channel proteins. In addition, fatty acid mediated alterations of ion channel activity could result from the effect of fatty acids on ion channel associated proteins.

To determine the mechanism of action of fatty acids on the activity of BK channels from rabbit pulmonary artery smooth muscle cells, all of the above mentioned mechanisms were considered. Most of the experiments described here were carried out using the patch-clamp technique and current recordings were performed in cell free, excised inside-out or outside-out membrane patches, in the absence of any added nucleotides and calcium.

As a first step towards understanding how fatty acids modulate BK channel activity, as well as the type of protein site with which fatty acids may be interacting, we determined the structural features of the fatty acid molecule that are required for channel modulation. To do this the effects of a range of fatty acids and other lipids on BK channel activity were examined. The features required for BK channel activation were found to be the negatively charged head group and a carbon chain of greater than eight carbons. We also found that positively charged lipids produced the opposite effect of negatively charged lipids, a decrease in BK channel activity. A similar chain length requirement was also necessary for channel inhibition by positively charged lipids; short chain compounds did not alter activity while those with fourteen carbons or greater decreased activity. The identification of these required structural features suggested that a specific interaction between the charge on the lipid head group is required for channel modulation by these lipids. The requirement for a chain length of greater than eight carbons also suggests that a hydrophobic interaction is necessary for these lipids to be effective modulators of this channel. In addition, the identification of these required structural features makes it unlikely that modulation of BK channel activity by these lipid compounds is a consequence of a perturbation of the lipid environment in which the channel resides.

Experiments were then carried out to determine whether modulation of BK channel activity by fatty acids and other charged lipids involved any of the following indirect mechanisms of action: 1) alterations in the concentration of calcium in the vicinity of the channel due to changes in membrane surface charge, or due to calcium stores attached to excised membrane patches, 2) alterations in the membrane electric field that the channel perceives due to changes in membrane surface charge and 3) changes in the activity of membrane bound protein kinases or protein phosphatases.

In experiments where high ionic strength solutions were used to shield membrane surface charge, fatty acids and other charged lipids were still able to modulate BK channel activity suggesting that fatty acids do not act through a mechanism involving surface charge. Experiments carried out in high concentrations of EGTA (20 mM) make it unlikely that calcium is involved in the modulation of BK channels by fatty acids and other lipids. The involvement of membrane bound kinases or phosphatases is also unlikely as fatty acids effectively modulated BK channel activity in the presence of staurosporin, a kinase inhibitor, and okadaic acid, a phosphatase inhibitor. The elimination of these indirect and non-specific suggests that fatty acids and charged lipids modulate BK channel activity by directly interacting with, either the channel protein itself, or some other channel associated protein.

To obtain further evidence that this indeed is the mechanism by which these lipids modulate BK channel activity; experiments were carried out to identify the site of action (i.e. side of the membrane) of both negatively and positively charged lipids. The negatively charged palmitoyl coenzyme A (PCoA) and a myristoylated positively charged peptide (myr-KPRPK), two compounds that are incapable of flipping across the bilayer, were used to identify the site of action of negatively and positively charged lipids. PCoA and myr-KPRPK produced their predicted effects of BK channel activation and suppression, respectively, only when they were applied to outside-out membrane patches. These experiments, therefore, support the contention that fatty acids and other charged lipids modulate BK channel activity by interacting with a site on the channel protein or a channel associated protein and that this site is found on the external membrane surface.

If the site responsible for channel modulation by fatty acids and other charged lipids is contained within the BK channel protein itself, other members of this family may also possess this site, and thus be modulated by fatty acids. Experiments were performed, therefore, to determine whether the BK cloned channels, mslo, hslo and bslo could also be modulated by fatty acids. These cloned channels were expressed in the Xenopus oocytes, and whole-cell currents were recorded using the two-electrode voltage clamp technique. The fatty acids myristic and arachidonic acid were able to increase whole-cell current of oocytes expressing all clone types. The modulation of these cloned channels by fatty acids did not appear to involve calcium, the BK β-subunit or a bioactive metabolite of arachidonic acid. Although all possible mechanisms of action were not addressed in this study, the results support the idea that the site of fatty acid interaction resides in the channel protein itself.

Taken together, therefore, these studies suggest that it is very likely that fatty acids and charged lipids modulate the activity of BK channels from smooth muscle cells of the rabbit pulmonary artery by directly interacting with an externally located site on the channel protein itself. The BK clones, mslo, hslo and bslo, are also modulated by fatty acids and it is likely that they share the same mechanism of action seen for BK channels from rabbit pulmonary artery smooth muscle cells.

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