Calcium-dependent block of P2X7 receptor channel function is allosteric

Among purinergic P2X receptor (P2XR) channels, the P2X7R exhibits the most complex gating kinetics; the binding of orthosteric agonists at the ectodomain induces a conformational change in the receptor complex that favors a gating transition from closed to open and dilated states. Bath Ca2+ affects P2X7R gating through a still uncharacterized mechanism: it could act by reducing the adenosine triphosphate4− (ATP4−) concentration (a form proposed to be the P2X7R orthosteric agonist), as an allosteric modulator, and/or by directly altering the selectivity of pore to cations. In this study, we combined biophysical and mathematical approaches to clarify the role of calcium in P2X7R gating. In naive receptors, bath calcium affected the activation permeability dynamics indirectly by decreasing the potency of orthosteric agonists in a concentration-dependent manner and independently of the concentrations of the free acid form of agonists and status of pannexin-1 (Panx1) channels. Bath calcium also facilitated the rates of receptor deactivation in a concentration-dependent manner but did not affect a progressive delay in receptor deactivation caused by repetitive agonist application. The effects of calcium on the kinetics of receptor deactivation were rapid and reversible. A438079, a potent orthosteric competitive antagonist, protected the rebinding effect of 2’(3′)-O-4-benzoylbenzoyl)ATP on the kinetics of current decay during the washout period, but in the presence of A438079, calcium also increased the rate of receptor deactivation. The corresponding kinetic (Markov state) model indicated that the decrease in binding affinity leads to a decrease in current amplitudes and facilitation of receptor deactivation, both in an extracellular calcium concentration–dependent manner expressed as a Hill function. The results indicate that calcium in physiological concentrations acts as a negative allosteric modulator of P2X7R by decreasing the affinity of receptors for orthosteric ligand agonists, but not antagonists, and not by affecting the permeability dynamics directly or indirectly through Panx1 channels. We expect these results to generalize to other P2XRs

TRPV1 channels are functionally coupled with BK(mSlo1) channels in rat dorsal root ganglion (DRG) neurons.

The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca(2+)). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca(2+) influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system

Miscellaneous properties of TRPV1/mSlo1 coupling.

<p>A, after treated with 2 μM TG for 5 minutes, the currents of TRPV1/mSlo1, co-expressed in HEK293 cells, and their time courses were obtained as described in Figure 2. The representative traces at -60 and 0 mV are shown in inset as indicated. B, with adding 5 mM EGTA into the intracellular solotion, the currents of TRPV1/mSlo1 and their time courses were obtained as described in Figure 2. The representative traces at -60 and 0 mV are shown in inset as indicated. C, the same as described in B, but for 10 mM BAPTA. The left inset shows the representative traces at -60 and 0 mV and the right inset show the mSlo1 currents activated by a set of voltages ranging from -60 to 160 mV.</p