Activation kinetics of single P2X receptors

After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X2, P2X4, and P2X7 receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level

Conduction properties of the M-channel in rat sympathetic neurons.

We have investigated the conduction properties of the M-channel in rat superior cervical ganglion neurons. Reversal potentials measured under bi-ionic conditions yielded a permeation sequence of Tl > K > Rb > Cs > NH4 > Na. Slope conductances gave a conductance sequence of K > Tl > NH4 > Rb > Cs. M-current was shown to exhibit a number of features atypical of potassium channels. First, the conduction of monovalent cations relative to K was very low. Second, the nature of the permeant ion did not affect the deactivation kinetics. Third, M-current did not exhibit anomalous mole-fraction behavior, a property suggestive of a multi-ion pore. Finally, external Ba, which is a blocker of M-current, showed a preferential block of outward current and had much less effect on inward current. The permeability sequence of the M-channel is very similar to other K-selective channels, implying a high degree of conservation in the selectivity filter. However, other conduction properties suggest that the pore structure outside of the selectivity filter is very different from previously cloned potassium channels

Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones

Whole-cell recordings were made from rat CA1 neurones in brain slices. When electrodes contained diazo-2 (2 mm) or dibromo BAPTA (1 mm) a large steady-state outward current (hundreds of picoamps) developed within 5 min of breakthrough at a VH of −60 mV. BAPTA itself (1 mm) caused qualitatively similar but smaller effects.The outward current was accompanied by increased conductance with a null potential close to the calculated K+ equilibrium potential (EK) of −110 mV. Development of outward current occurred concurrently with progressive loss of slow AHP tail current (IsAHP) evoked by brief depolarizations. The peak latency of IsAHP increased during the onset of chelator action.The persistent outward current was reversibly inhibited by noradrenaline (10 μm) or isoprenaline (2–5 μm), and completely prevented by 8-bromoadenosine 3′,5′ cyclic monophosphate (8-Br cAMP; 100 μm) or QX-314 (10 mm) in recording electrodes. After development of outward current, diazo-2 photolysis caused inward current and decreased conductance. Both flash- and noradrenergic-sensitive responses were inwardly rectifying outward currents with null potentials close to EK.The outward current induced by dibromo BAPTA was not blocked by internal EGTA (10 mm). However, experiments incorporating Ca2+ influx or Ca2+ loading of the buffer indicate that Ca2+ facilitated the outward current.The outward currents induced by dibromo BAPTA or diazo-2 were not associated with significant changes in resting [Ca2+]i. Regions of the cell contributing to the outward current were deduced from measurements of fura-2 diffusion. These were compared with regions of [Ca2+]i elevation during IsAHP.These results are consistent with the hypothesis that the BAPTA series Ca2+ buffers can activate those Ca2+-activated K+ channels that underlie the slow AHP, without the predicted elevation of bulk [Ca2+]i. Therefore these results cannot be interpreted solely in terms of Ca2+ concentration changes, although the observations illustrate a novel, investigative role for these compounds in the study of Ca2+-dependent processes

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

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