**1. Introduction**

Acid-sensing ion channels (ASICs) are cation channels from the degenerin/epithelial sodium channel (DEG/ENaC) superfamily. They are activated by fast acidification of the media, while prolonged exposure leads to their desensitization. There are five paralogous genes in this group, with the expression products of *ASIC1*, *2*, and *3* forming functional trimeric channels. *ASIC1* and *ASIC2* are predominantly expressed in the central nervous system, whereas *ASIC3* is more common in the peripheral nervous system [1]. The functions they fulfill also vary. ASIC1 and ASIC2 have been shown to contribute to the excitatory postsynaptic currents [2] and synaptic plasticity [3], and are also involved in the pathologic processes in stroke and ischemia [4,5]. On the other hand, ASIC3 is typically associated with peripheral nociception [6]. Another important difference is that ASIC3 channels, unlike other ASICs, do not fully desensitize during prolonged activation, supporting a significant sustained current [7].

The involvement of ASICs, in particular ASIC3, in the perception of pain has been firmly established in a number of studies (for review see [8]). The use of ASIC inhibitors in rats and humans

was shown to alleviate cutaneous pain and hyperalgesia [9–11]. Surprisingly, knockout of the *ASIC3* gene in mice did not lead to a loss or significant decrease of their pain responses compared to wild type [12]. In fact, in the study of Kang et al. [13], triple knockout (for *ASIC1a*, *2* and *3* genes) mice showed enhanced pain sensitivity. This phenomenon can potentially be explained by the different roles of the ASIC channels in different species or particular levels and by specific details of their expression [14]. Large acidification-evoked currents were also shown in cardiac afferents, where they propagated cardiac pain and angina [15]. Characteristics of those currents are closely matched by heteromeric ASIC3/ASIC2b channels [16]. Other pain-associated conditions are also mediated by ASIC3, such as migraines [17], osteoarthritis [18], and muscle inflammation [19].

Given the importance of their role and the potential of new functions' discovery, it is not surprising that ASIC pharmacology receives quite a lot of attention [20]. The pioneering paper by Waldmann et al. [21] described the action of amiloride, a common modulator of ENaC channels, which was found to be a low-affinity inhibitor of ASICs. Focusing on ASIC3 for the purposes of this work, there are several groups of drugs to be noted. The abovementioned amiloride inhibits peak currents of ASIC3 but does not affect the window current. Even more interestingly, in high concentrations it is capable of inducing said window current by itself, without acidification [22]. 2-Guanidine-4-methylquinazoline (GMQ) was also originally described as an ASIC3 modulator, although, unlike its predecessor, it has a potentiating effect. Like amiloride, GMQ can also evoke ASIC3 currents in neutral pH [23]. Later it was found that GMQ and its derivatives can also modulate ASIC1a [24].

Several endogenous compounds were shown to potentiate ASIC3 currents, including FMRFamides and related peptides [25], agmatine [26], and serotonin [27], with the last one only affecting the sustained component of the response. Agmatine was also able to activate the channels directly.

On the other hand, toxins mostly display inhibitory action on ASIC3. A number of sea anemone toxins, such as APETx2 [28] and Ugr 9-1 [29], inhibit both peak and window currents in ASIC3. MitTx [30], which locks the channel in the open state, also works on ASIC3 but in significantly higher concentrations than on the other subunits.

In our research [31] we focused our attention on a group of small-molecule ligands we collectively called *hydrophobic monoamines*. Despite their structural simplicity, further investigations revealed quite complex effects that they can induce on ASIC channels [32]. We found that they can block the channel pore, affect the activation curve in either direction, and shift the desensitization curve to more acidic values, often with several effects observed for a single compound. Additionally, through this line of investigation a potential physiological modulator of ASICs (i.e., histamine) was discovered [33]. Its effects were specific to ASIC1a homomers. However, outside of initial assessment [31], the action of monoamines on ASIC3 was never studied. Thus, in the present work we attempted to elucidate the mechanisms of action of hydrophobic monoamines and their guanidine analogs on ASIC3 channels. Other compounds that were found to affect ASIC1a and/or ASIC2a, such as some antidepressants [34] and histamine receptor agonists [35], were also included in the study.

#### **2. Results**

#### *2.1. Drug Selection*

Several groups of compounds were selected for the present study. The IEM line ofcompounds was originally designed as glutamate receptor agonists [36,37]. Their activity on ASIC channels was subsequently shown by our group [31,32]. Memantine [38] and 9-aminoacridine [39] also affect glutamate receptors as well as ASICs [31]. Other drugs included long-established antidepressants amitriptyline and tianeptine [40,41] and histamine receptor modulators imetit, dimaprit, and thioperamide [42,43]; their effects on ASICs were recently established in [34] and [35], respectively. It is important to note that in previous studies only the effects on ASIC1a and ASIC2a were examined, with ASIC3 covered very briefly in [31].
