Unoprostone activation of BK (KCa1.1) channel splice variants

Abstract

This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.

Keywords: BK channels; Ca(2+)-dependence; KCNMA1; Rescula®; Single channels; Unoprostone.

Figure 1. A schematic diagram of the two BK channel constructs, rSlo(27) and rSlo(0)

A construct for muBKa1 (mouse) was truncated at position 953. Then the c terminus of rBKa1 was added to the truncated form to produce rSlo(0). In the second construct, bases corresponding to 27 amino acids were added before once again adding the c terminus of rBKa1 to produce rSlo(27). In this figure the sequence between the N-terminus and 9^th alpha helical domain are identical, but the 27 amino acid region immediately prior to the calcium “bowl” are present in one construct and not the other. The sequence of the calcium “bowl” and the subsequent sequence to the C-terminus are identical.

Figure 2. The response to calcium of rSlo(0) and rSlo(27) splice variants is the same

The response of the two splice variants to intracellular calcium was indistinguishable in HEK293 cells which stably express rSlo(27) or rSlo(0). In excised patches depolarized to +40 mV, increasing calcium produced a statistically significant increase in open probability for all calcium concentrations (p<0.5), but there is no statistically significant difference in channel open probability between rSlo(27) and rSlo(0) at any concentration of calcium (each bar is the mean ± s.d.of 4 separate experiments).

Figure 3. Response of whole cell BK currents to unoprostone

Whole cell currents in response to the voltage steps shown in panel A. Currents from HEK cells transfected with rSlo(27) internally perfused with 1μM Ca²⁺ (panel B) and the same cells with 10 nM unoprostone in the bath (panel C). I-V plots from the cells that are unoprostone-treated or untreated cells are shown for rSlo(27) (panel D) and rSlo(0) (panel E). Each point in the I-V relationships represents the mean ± s.e.of 3 separate experiments.

Figure 4. Response of BK channel variants to unoprostone

In excised patches, unoprostone increases the open probability of rSlo(27) (panel A), but not rSlo(0) (panel B). Panel C summarizes the effect of unoprostone on open probability of the two splice variants (from means ± s.e.of 3 separate analyses of 1 minute continuous recording). With the patch depolarized to +40 mV and 50 nM Ca²⁺ in the bath, increasing concentrations of unoprostone increases open probability of rSlo(27) channels, but has little if any effect on rSlo(0). This is not because rSlo(0) is inactive (see Fig. 2) since when bath Ca²⁺ is increased to 500 nM (bottom trace), rSlo(0) activity is strongly increased.

Figure 5. Unoprostone does not alter the voltage dependence of BK channels

Although the open probability of BK channels is uniformly increased by unoprostone the slope of the Po vs voltage relationship is not significantly different in the presence of unoprostone (44 ± 4.8, 48 ± 5.2, and 46 ± 4.6 mV per 10 fold change in Po at 10, 1, and 0 nM unoprostone, respectively). Points are the mean ± s.e.of 3 separate experiments.

Figure 6. Open probability of BK channels vs calcium concentration in the presence of different unoprostone concentrations

We examined BK channel activity in excised patches at +60 mV with final concentrations of calcium of 0.01, 0.1, 1, 2, 5, 10, 20, 50, 100, 500, or 1000 nM. To each of these patches we sequentially applied 0, 0.1, 0.2, 1, 2, 5, 10, 20, or 100 nM unoprostone. The principal effect of unoprostone is to shift the calcium-concentration response curve to the left; i.e., unoprostone makes the channel more sensitive to calcium. The half-maximal stimulation of the sensitive BK channels by Ca²⁺ was shifted from 3.4 ± 0.017 nM to 0.81 ± .0058 nM in the presence of 10 nM unoprostone. Each point represents the mean ± s.e.of 3 separate experiments.

Figure 7. Unoprostone does not activate BK channels in the absence of calcium

We examined BK channel activity in excised patches at +60 mV with final concentrations of 0, 0.1, 0.2, 1, 2, 5, 10, 20, or 100 nM unoprostone. To each of these patches we sequentially applied calcium of 0.01, 0.1, 1, 2, 5, 10, 20, 50, 100, 500, or 1000 nM. At low calcium concentrations, there was little activation of BK channels by unoprostone imply in that the principal effect of unoprostone was to increase calcium binding affinity. Each point represents the mean ± s.e.of 3 separate experiments.

Figure 8. A 3-D representation of the relationship between unoprostone, calcium, and open probability

This figure represents all the data of figures 6 and 7 collected together and fit with to the model given in the discussion.

Figure 9. Unoprostone activates BK channels in cell-attached patches

The single channel record at the top is a long continuous record from a cell-attached recording from a HEK cell stably transfected with rSlo(27) to which sequentially larger concentrations of unoprostone are added. The patches were depolarized to +60 mV. Sections of the record at A, B, C, and D are expanded in the traces below to show individual channel events. The single channel record at the bottom is a long continuous record from a cell-attached recording from a HEK cell stably transfected with rSlo(0) to which sequentially larger concentrations of unoprostone are added. Sections of the record at A, B, C, and D are expanded in the traces below to show individual channel events. This experiments show that under resting conditions with an intact cell, unoprostone applied extracellulary can activate rSlo(27) BK channels, but not rSlo(0) channels. To the right is the summary data showing the relationship of unoprostone concentration to BK channel open probability (Each point represents the mean ± s.e. of 3 separate experiments). When applied to the extracellular surface about 10 times as much unoprostone is necessary to activate channels than in excised patches.

Figure 10. RNA from human trabecular meshwork cells contains a BK channel splice variant that corresponds to rSlo(27)

Total RNA from Human Trabecular Meshwork Cells was used to prepare cDNA by reverse transcription. Two sets of PCR primers specific for human BK channels were designed based on published BK channel sequences (NCBI) to correspond to regions that were completely conserved in all published human sequences and are also present in rSlo(0) and rSlo(27). The first set of primers amplified a region from just N-terminal to the eighth alpha helical domain (see Fig. 1) to a region just before the ninth alpha helical region in the extended intracellular domain. The second set of primers amplified a region that included the “calcium bowl”, the tenth alpha helical region, and the splice site at which 27 amino acids were inserted in rSlo(27). We amplified partial clones with these primer pairs and the PCR products were run on a 0.8% gel. Two bands of similar molecular weight were obtained from the first set of primers and a single band was obtained from the second set (Fig. 10A).. The DNA was cloned into PCR2.1-TOPO (Invitrogen). 13 clones from the first set of amplimers and 5 clones from the second set were sequenced. All five clones from the second set of primers had the same sequence (Fig. 10C) containing a region completely homologous to rSlo(27) implying that BK channels in the cells from which this RNA was derived should be sensitive to unoprostone. Interestingly, 1 out of 13 clones amplified by the first set of primers was a splice variant containing a 29 amino acid insert (Fig. 10B).