Functional expression of SK channels in murine detrusor PDGFR+ cells

Abstract

We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFRα(+) cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFRα(+) cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFRα(+)/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFRα(+) cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFRα(+) cells in comparison to SMCs. αSlo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFRα(+) cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca(2+) currents in SMCs, but inward current conductances were not activated in PDGFRα(+) cells under the same conditions. PDGFRα(+) cells displayed spontaneous transient outward currents (STOCs) at potentials positive to -60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFRα(+) cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca(2+)) in PDGFRα(+) cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFRα(+) cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials.

Figure 1. End product gel and quantitative PCR of Ca²⁺-activated K⁺ channels in unsorted, sorted SMC and sorted PDGFRα⁺ cells

A, sorted PDGFRα⁺ cells were minimally positive for Mhc (smooth muscle cell marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 and αslo were detected in sorted PDGFRα⁺ cells. B, Pdgfra was highly enriched (12 fold vs. unsorted cell) in PDGFRα⁺/eGFP cells. SK3 was dominantly expressed in PDGFRα⁺ cells in comparison to SMC and unsorted cells. αslo (BK marker) was more highly expressed in SMC. Relative expressions of all transcripts were normalized to Gapdh.

Figure 2. Immunohistochemistry of PDGFRα and SK3 channels in murine detrusor

Whole-mount of the tunica muscularis in murine detrusor, displays immunoreactivity of PDGFRα⁺ cells and SK3 channels. These proteins were co-localized in PDGFRα⁺ cells in detrusor muscles.

Figure 3. Comparison of current–voltage relationships of a smooth muscle cell (SMC) and a PDGFRα⁺ cell

SMCs and PDGFRα⁺ cells expressed voltage-dependent outward currents (A and B). Depolarization evoked voltage-dependent Ca²⁺ currents in SMCs (C), but no inward current conductances were apparent under the same conditions in PDGFRα⁺ cells (D, see arrow). E and F demonstrate the summarized data of outward currents (E) and inward currents (F) from both types of cells.

Figure 4. Spontaneous transient outward currents (STOCs) in PDGFRα⁺ cells

A, STOCs were observed in PDGFRα⁺ cells dialyzed with solutions containing 200 nm[Ca²⁺]_i,. Green arrow heads denote ramp depolarizations from −80 to + 80 mV. B, expanded time scale from panel A (red dotted box). a and b denote ramp depolarizations. C, current responses to ramp depolarizations before (a) and during a STOC (b). Note reversal potential, lack of voltage-dependence at negative potentials, and inward rectification at positive potentials of current during STOC. These are classic signatures of SK currents (Soh & Park, 2001, 2002). Lower trace shows voltage-ramp protocol. D, apamin inhibited STOCs. Cell held at −40 mV. Green arrow heads denote ramp depolarizations. Inset shows current-voltage relationship during a STOC before (a) and after (b) apamin. E, iberiotoxin did not inhibit STOCs. Cell held at −40 mV. Inset shows current-voltage relationship during STOCs before (a) and after (b) Iberiotoxin. Lower traces in D and E insets denote voltage-ramp protocols.

Figure 5. Effects of CyPPA and SKA-31 on membrane potential and SK currents in PDGFRα⁺ cells

A, CyPPA (10 μm) induced membrane hyperpolarization under current clamp (I= 0, blue dot line). In the same cell, CyPPA activated outward current (current above red dotted line) at a holding potential of −40 mV under voltage-clamp mode (V-C). B, current responses to ramp-depolarizations before (a) and during (b) CyPPA. Inset denotes voltage-protocol. C, SKA-31(10 μm) induced membrane hyperpolarization under current clamp (I= 0, blue dot line). In the same cell, SKA-31 activated outward current (current above red dotted line) at a holding potential of −40 mV under voltage-clamp mode (V-C). D, current responses to ramp-depolarizations before (a) and during (b) SKA-31. Inset denotes voltage-protocol.

Figure 6. Single channel recordings from membrane patches excised from PDGFRα⁺ cells

A, representative traces displayed single channel currents at −20 mV to +40 mV. Solid line denotes channel close and dotted line denotes channel opening. B, corresponding amplitude histogram from panel A. no of obs; numbers of observation. C, an increase in cytosolic [Ca²⁺] from 10nM to 300nM under excised patch increased open probability of SK channels. Lower panel display the expanded time scale from red box in upper panel. c; channel close, o; channel open, o1–o3; number of channel opening. D, plot of [Ca²⁺]vs. normalized current at 0 mV during ramp depolarization in 10⁻⁶ m[Ca²⁺] were constructed to describe Ca²⁺ sensitivity.

Figure 7. CyPPA and SKA-31 had no effect on smooth muscle cells (SMCs) at physiological potentials

A, CyPPA had no resolvable effect on membrane potentials (I= 0, blue dotted line) and currents (voltage-clamp at −40 mV, red dotted line). B, SKA-31 did not cause hyperpolarization (I= 0, blue dotted line) or activate outward currents at a holding potential of −40 mV (voltage-clamp, red dotted line).