The Journal of general physiology
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The term excitation-coupled Ca(2+) entry (ECCE) designates the entry of extracellular Ca(2+) into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca(2+) entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 microM), and is permeable to Mn(2+). Here, we have examined the effects of these agents on the L-type Ca(2+) current conducted via the DHPR. ⋯ Like ECCE, the L-type Ca(2+) channel displays permeability to Mn(2+) in the absence of external Ca(2+) and produces a Ca(2+) current that persists during prolonged ( approximately 10-second) depolarization. This current appears to contribute to the Ca(2+) transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca(2+); (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR alpha(1S) pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca(2+)-free solution and were unaffected by Ca(2+) removal; and (3) after block of SR Ca(2+) release by 200 microM ryanodine, normal myotubes still displayed a large Ca(2+) transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca(2+) entry attributed to ECCE.
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Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. ⋯ Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.
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Low voltage-activated (LVA) T-type Ca(2+) (I(Ca)T) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and I(Ca)T currents. ⋯ CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aalpha/beta-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.
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Comparative Study
Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia.
A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-mum periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to >or=7 microm are poorly understood. Using bumetanide as an inhibitor of Cl- secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated "blending" of both Cl- secretion and Na+ absorption must occur to effect ASL volume homeostasis. ⋯ ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl- secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume.