Measurement of relative Ca2+ permeability during sustained activation of TRPV1 receptors

Abstract

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.

Myrcene and terpene regulation of TRPV1

“…Myrcene currents shared similar properties to those evoked by capsaicin, especially in their dependence on internal and external calcium levels. One distinction, however, was the lack of transition to the pore-dilated, non-rectifying, channel open state that is associated with capsaicin-induced currents [53][54][55]. Interestingly we also note this lack of pore dilation in TRPV1 responses to CBD, CBN, and CBD [46].…”

“…TRPV1 is a two state channel, exhibiting rapid pore dilation after activation in response to Capsaicin [53][54][55]. This pore-dilation results in a loss of selectivity, rendering the channel permeant to large cations (e.g., NMDG) and in this state, the I/V relationship becomes highly linearized and reverses at close to 0 mV potentials.…”

Direct Anandamide Activation of TRPV1 Produces Divergent Calcium and Current Responses

“…One major critique of these experiments is the likelihood of significant ion accumulation intracellularly that may be sufficient to shift the relevant equilibrium potentials of permeant ions (Li et al, 2015; Samways et al, 2016). Here we report in native TRPV1 expressing neurons differential sensitivity to extracellular calcium between CAP and AEA activation of TRPV1.…”

TRPV1 is a component of the atrial natriuretic signaling complex, and using orally delivered antagonists, presents a valid therapeutic target in the longitudinal reversal and treatment of cardiac hypertrophy and heart failure

“…The transient receptor potential cation channel subfamily V, member 1 (TRPV1) is an ionotropic nonselective cation channel. It was initially identified in peripheral sensory neurons and later found to be widespread in the cardiovascular system [9][10][11][12][13][14]. TRPV1 appears to be important in the heart by virtue of the fact that its genetic knockout or pharmacological inhibition rescues cardiac hypertrophy in the mouse [18,19,25].…”

“…New targets are needed because extant therapies are not adequately addressing these needs. The transient receptor potential cation channel subfamily V, member 1 (TRPV1) is an ionotropic nonselective cation channel, initially identified in peripheral sensory neurons and found widespread in the cardiovascular system [9][10][11][12][13][14]. Studies have implicated the role of endogenous activator anandamide (ANA) in multiple cardiovascular diseases, such as myocardial ischemia reperfusion injury and hypertension [15,16].…”