Background and purpose: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta.

Arterial interstitial cells of Cajal (ICC)‐like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 μm), sometimes branching, processes with lengths up to ≈60 μm were isolated enzymatically from 1st to 7th order branches of guinea‐pig mesenteric artery. Some of the processes of AIL cells were growing (average speed ≈0.15 μm min−1) and their growth was blocked by 10 μM latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F‐actin, showed the presence of F‐actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 μM nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 mM 4‐aminopyridine and/or 100 nM iberiotoxin but unaffected by 10 nM dendrotoxin‐K. Imaging of intracellular ionised calcium with fluo‐4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in ≈28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long‐lasting transient outward currents, which occurred at potentials positive to −40 mV. Unlike myocytes, AIL cells did not contract in response to 1 mM caffeine or 5 μM noradrenaline, although they responded with a [Ca2+]i increase. The segments of intact arteries did not stain for c‐kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC‐like cells is demonstrated for the first time in the media of resistance arteries.

Previously we have described a constitutively active, Ca2+ -permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd 3+ , La 3+ and Ca 2+ inhibited spontaneous channel activity in outside-out patches with IC 50 values of 6.8 μM, 25 nM, 1.5 μM and 0.124 mM, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.