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Flufenamic acid has been known since the 1960s to have anti-inflammatory properties attributable to the reduction of prostaglandin synthesis. Thirty years later, flufenamic acid appeared to be an ion channel modulator. Thus, while its use in medicine diminished, its use in ionic channel research expanded. Flufenamic acid commonly not only affects non-selective cation channels and chloride channels, but also modulates potassium, calcium and sodium channels with effective concentrations ranging from 10(-6)M in TRPM4 channel inhibition to 10(-3)M in two-pore outwardly rectifying potassium channel activation. Because flufenamic acid effects develop and reverse rapidly, it is a convenient and widely used tool. However, given the broad spectrum of its targets, experimental results have to be interpreted cautiously. Here we provide an overview of ion channels targeted by flufenamic acid to aid in interpreting its effects at the molecular, cellular, and system levels. If it is used with good practices, flufenamic acid remains a useful tool for ion channel research. Understanding the targets of FFA may help reevaluate its physiological impacts and revive interest in its therapeutic potential., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This chapter describes methods to investigate mammalian cardiac channel properties at the single-channel level. Cell isolation is performed from adult heart by enzymatic digestion using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. Also, isolation of human atrial cardiomyocytes is described. In freshly isolated cells or cells maintained in primary culture, the single-channel variants of the patch-clamp technique (cell-attached, inside-out, and outside-out) are used to investigate channel properties. Biophysical properties such as conductance and ionic selectivity are determined. Also, regulations by extracellular and intracellular mechanisms are investigated. To illustrate the study, the author provides an example by the characterization of a calcium-activated nonselective cation channel (TRPM4).

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i-sensitive currents: the Na(+)-Ca2+ exchange current, a Ca(2+)-activated chloride current and a Ca(2+)-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca(2+)-activated non-selective cation channel (NSC(Ca)) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I-V relationship with a conductance of 19 +/- 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 microM. Channel activity was reduced in the presence of 0.5 mM ATP or 10 microM glibenclamide on the cytoplasmic side to 22.1 +/- 16.8 and 28.5 +/- 8.6%, respectively, of control. It was also inhibited by 0.1 mM flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC(Ca) channel is a serious candidate to support the delayed after-depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

Cardiac hypertrophy is associated with electrical activity modifications, including sustained depolarization, that lead to a propensity for arrhythmias. The ionic currents underlying the sustained depolarization are not well defined. Similar modifications were reported on adult rat cardiomyocytes in primary culture undergoing dedifferentiation. Using the single-channel measurements on these cells, we identified the appearance of a Ca2+-activated nonselective cation channel (NSCCa) during the dedifferentiation process. In excised inside-out patches the channel presented a linear I/V relationship with a conductance of 26.5 pS. It was equally selective for Na+ and K+ and impermeable to Cl- and Ca2+ ions. The open probability increased with depolarization and with rise in intracellular calcium concentration. The channel activity was reduced by intracellular ATP and suppressed by flufenamic acid. Channel detection increased after incubation with a purinergic receptor agonist (ATPgS) or a PKC activator (PMA). Furthermore, occurrence of the channel developed during the culture. Absent at one day in vitro (d.i.v.), channel activity was present in 5, 46, 27 and 19% of patches after 4, 7, 14 and 21 d.i.v., respectively. We suggest that the channel may be associated with pro-arrhythmic signaling, in particular during the release of transmitters from autonomic nerve endings in the hypertrophied hearts.

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (Po), giving a bell-shaped curve peaking at pH 6.8/7.0: Po at pH 6.0 was 11 +/- 6% of Po at pH 7.2 and 32 +/- 7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K(+)-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5 +/- 3.1 pS and 17.3 +/- 1.8 pS, respectively (n = 4). In contrast, cell-attached patches were usually inactive when a Na(+)-rich solution filled the patch pipette. However, another type of channel with a conductance of 20-30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1 +/- 1.2 pS (n = 17), excluded anions (PCl/PNa = 0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa = 1.5; PK/PNa = 0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa = 0.09; PBa/PNa approximately 0). The cation channel was moderately voltage-dependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 mumol/l-0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition of Po at 1.2 mumol/l AMP. As in other cell models, 3',5'-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.

The phenanthrene-derivative 9-phenanthrol is a recently identified inhibitor of the transient receptor potential melastatin ( TRPM) 4 channel, a Ca 2+-activated non-selective cation channel whose mechanism of action remains to be determined. Subsequent studies performed on other ion channels confirm the specificity of the drug for TRPM4. In addition, 9-phenanthrol modulates a variety of physiological processes through TRPM4 current inhibition and thus exerts beneficial effects in several pathological conditions. 9-Phenanthrol modulates smooth muscle contraction in bladder and cerebral arteries, affects spontaneous activity in neurons and in the heart, and reduces lipopolysaccharide-induced cell death. Among promising potential applications, 9-phenanthrol exerts cardioprotective effects against ischaemia-reperfusion injuries and reduces ischaemic stroke injuries. In addition to reviewing the biophysical effects of 9-phenanthrol, here we present information about its appropriate use in physiological studies and possible clinical applications. [ABSTRACT FROM AUTHOR]