Chloride Single Channel Activity in Freshly‐Isolated Guinea Pig Detrusor Smooth Muscle Cells

Abstract

Ion channels in detrusor smooth muscle (DSM) control excitability and contractility of the urinary bladder. Several ion channel types have already been detected and characterized in DSM cells, especially voltage‐gated cationic channels. However, little is known about Cl− channels in DSM cells. Specifically, it is unknown what Cl− channel subtypes are expressed in the plasma membrane of DSM cells and how they control DSM cell excitability. Earlier molecular and fluorometric studies suggested the presence of Ca2+‐activated Cl− channels in DSM cells. Our Agilent Human GE 4x44K v2 microarray data analysis on single DSM cell mRNA detected a number of candidates underlying Cl− conductance in DSM cells including plasmalemmal TMEM16A/ANO1, CLCN3, CLCN6, and CLCN7. Thus, detailed biophysical analyses are needed to characterize these Cl− channels. Here, guinea pig DSM cells were freshly isolated by an enzymatic digestion method with papain and collagenase type II, plated at the glass bottom of a recording chamber, and electrophysiologically tested within 6 hours after the isolation. Inside‐out single channel patch clamp recording technique was used to detect electrical activity of Clchannels in the plasma membrane of isolated DSM cells. To separate Cl− from cationic currents, all experiments were done using asymmetrical Cl− and symmetrical Na+ solutions. The bath/intracellular solution contained (in mM): 110 Na‐glutamate, 5 NaCl, 60 mannitol, 10 HEPES, 1 CaCl2, 1 CoCl2, 0.0005 phorbol 12‐myristate 13‐acetate, pH 7.2; the pipette/extracellular contained (in mM): 110 NaCl, 1.5 MgCl2, 2 CaCl2, 60 mannitol, 10 HEPES, 0.001 paxilline, 0.01 nifedipine, pH 7.4. Cl− single channel activity detection frequency varied from zero to 26.7 % within a single DSM cell preparation with an average frequency for detection of 7.4 % (13 out of 175 patches, 8 guinea pigs). Cl− channels displayed unique gating with multiple subconductive states and a fully opened single‐channel conductance in a range from 140 pS to 193 pS with an average value of 163 ± 15 pS (12 patches, 8 guinea pigs, mean ± SD). A linear fitting of single channel current amplitude – membrane potential relationships yielded a reversal potential of − 41.3 ± 5.5 mV (12 patches, 8 guinea pigs, mean ± SD) for this Cl− channel, close to the theoretical ECl of − 65 mV and confirming that this channel is permeable to Cl−. The Cl− channel demonstrated strong voltage‐dependence of activation with virtually no activity below the measured reversal potential and always robust prolonged openings at depolarizing voltages. The channel had similar gating when exposed to the intracellular Ca2+‐free (buffered with 5 mM EGTA in nominal Ca2+) and 1 mM Ca2+ solutions. The Cl− channel was not blocked by 9‐phenanthrol (30 μM), a transient receptor potential melastatin 4 channel inhibitor that also has been reported to block the activity of TMEM16A Ca2+‐activated Cl− channels in vascular smooth muscle cells. In conclusion, our data reveal for the first time the presence of Ca2+‐insensitive voltage‐dependent Cl− channel in guinea pig DSM cells at the single channel level. This channel may be a key regulator of DSM excitability.Support or Funding InformationSupported by NIH R01DK106964 to Georgi V. Petkov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.