TEA- and apamin-resistant KCa channels in guinea-pig myenteric neurons: slow AHP channels

摘要

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K⁺ channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (Po) of TEA-resistant K⁺ channels following action potentials. The Po increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca²⁺-activated K⁺ (KCa) channels were present in inside-out patches excised from AH neurons. The Po of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca²⁺] from < 10 nm to either 500 nm or 10 μm. Po was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant KCa channels measured approximately 60 pS under symmetric K⁺ concentrations between −60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K⁺], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant KCa channels by internal Ca²⁺ in excised patches was not reversed by washing out the Ca²⁺-containing solution and replacing it with nominally Ca²⁺-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant KCa channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca²⁺ dependence of the activation of TEA- and apamin-resistant KCa channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca²⁺ to about 7 ms in the presence of submicromolar cytoplasmic Ca²⁺. The Ca²⁺-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca²⁺-activated K⁺ channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.