HISTORIA DE LA DETECCI ´ ON DE GRBs

K+ channels are by far the largest and the most diverse group of ion channels. They are expressed in almost every cell type in which they play various physiological roles (129). Between 70 and 80 genes are known to code for K+ channel proteins (129). In the DSM, the physiological role of K+ channels is to regulate muscle contraction and relaxation (130). This function is achieved by a precise regulation of intracellular Ca2+ concentration which is controlled by Ca2+ entry via voltage-gated Ca2+ (Cav) channels

(130). In general, K+ channels’ opening causes cell membrane hyperpolarization, interruption of Ca2+ entry via Cav channels, and DSM relaxation (129, 130). By contrast,

K+ channels’ inhibition causes membrane depolarization, increase of Ca2+ entry via Cav,

and DSM contraction (129, 130). Structurally, all K+ channels form a pore-loop and can have 2 to 8 transmembrane (TM) domains (Fig. 1.5.1). K+ channels are classified into four major families including voltage-gated (Kv), Ca2+-activated (KCa), inward rectifying

Figure 1.5.1. Illustration of the transmembrane architecture and subunit stoichiometry of the K+ channel types expressed in detrusor smooth muscle cells. Kir channels (represented by KATP channel) have the simplest K+ channel

structure, with two transmembrane segments (S) connected by a pore loop. Four such subunits form a functional tetrameric channel pore. K2P channels form a tetrameric pore structure from two subunits each containing two pore

loops. Kv channel subunits have six transmembrane segments with a voltage sensor in the S4 transmembrane domain. BK channels consist of four pore-forming α-subunits and the four regulatory β1 or β4 subunits. Abbreviations: BK channels, large-conductance voltage-activated and Ca2+-activated K+ channels; K2P, two-pore-domain K

+

channels; KATP, channels, inward-rectifying ATP-sensitive K

+

channels; Kir channels, inward-rectifying K +

channels; Kv channels, voltage-gated K+ channels. Reprinted from Petkov (2012) with permission.

Voltage-gated K+ (Kv) channels form the most diverse family of K+ channels and are encoded by more than 40 genes (130). Kv channels are known to participate in the repolarization phase of the action potential and in maintaining the cell’s resting membrane potential (Fig. 1.5.2) (56, 60, 74, 77, 155). Kv channels are classified into 12 subfamilies (Kv1 – Kv12) based on the amino-acid sequence homology. Each Kv gene encodes a pore-forming α-subunit protein which assembles into tetramer to form a functional channel (130). While Kv1, Kv2, Kv3, Kv4, Kv7, Kv10, Kv11, and Kv12 channels can form their own homotetramers, Kv5, Kv6, Kv8, and Kv9 channels cannot but rather form heterotetramers by assembling with the Kv2 channel α-subunit (130). Despite, the significant number of Kv channel genes identified in the human genome, only a small fraction of Kv channels has been studied in the DSM suggesting that more work is needed to determine if these channels can potentially regulate DSM contraction and relaxation.

Ca2+-activated K+ (KCa) channel family has five members (KCa1.1, KCa2.1 KCa2.2,

KCa2.3, and KCa3.1) which can be classified based on the single channel conductance

(130). The large conductance voltage-activated and Ca2+-activated K+ (KCa1.1 or BK)

channel is also known as big-K or maxi-K because of its high single channel conductance (100 – 250 pS) (130). The BK channel can be activated by both Ca2+ and voltage and is highly expressed in the DSM where it plays a role in maintaining the resting membrane potential and securing the repolarization phase of the action potential (60, 61, 67, 75, 80, 130). The small conductance Ca2+-activated K+ (SK) channels have a single channel conductance ranging from 5 to 20 pS (129, 130). There are 3 types of SK channels which

include SK1 (KCa2.1), SK2 (KCa2.2), and SK3 (KCa2.3) channels (129, 130). SK channels

are voltage-insensitive and are regulated by Ca2+ and calmodulin (106). In the DSM, they are suggested to contribute to the afterhyperpolarization phase of the action potential (Fig. 1.5.2) (45, 56). The intermediate-conductance Ca2+-activated K+ (KCa3.1 or IK or

SK4) channel has a single channel conductance ranging from 20 to 80 pS (129, 130). IK channels are activated by Ca2+ and insensitive to voltage changes (129, 130). Their role in the DSM remains unclear since most studies suggest that pharmacological modulation of these channels has no effects on DSM excitability and contractility (2, 127).

Inward-rectifying ATP-sensitive K+ (Kir) channels induce high amplitude of inward K+

currents during the hyperpolarization phase of the action potential (129, 130). In the DSM, the Kir channels are represented by the Kir6 (KATP) channel which has a single

channel conductance ranging from 11 to 12 pS (86, 87). These channels are activated by both low intracellular ATP and high ADP levels (129, 130). Whether KATP channels are

physiologically relevant in the DSM remains controversial as evidence of their physiological role or not are conflicting (Fig. 1.5.2). Some studies argues in favor of KATP channels’ ability to regulate DSM contractility and excitability (45, 58, 86, 87, 133,

141) while others suggest that activation of KATP channel with glibenclamide, a specific

KATP channel inhibitor has not effects on DSM contractility and excitability (37, 133,

141).

Two-pore-domain K+ (K2P) channels has 15 members including (K2P1 – K2P15) which are

mostly known for their role in stretch, oxygen, pH and temperature sensing (12, 129, 130). They play a role in maintaining the cell’s resting membrane potential (Fig. 1.5.2). In the DSM, knowledge about K2P channels is still at its infancy. The few studies which

investigated K2P channels subtypes (TASK-1 and TASK-2) in the DSM suggest that these

channels play an important role in stabilizing the DSM cell’s resting membrane potential (12).

Figure 1.5.2. Schematic illustration of the detrusor smooth muscle action potential and the roles of various K+ channels in determining resting membrane potential and action potential. BK, Kv, K2P, and probably KATP

channels determine the resting membrane potential. BK and Kv channels contribute to the initial repolarization phase of the action potential. SK and Kv channels contribute to the prolonged after-hyperpolarization phase of the action potential. Abbreviations: BK channels, large-conductance voltage-activated and Ca2+-activated K+ channels; Cav, voltage-gated Ca2+ channels; K2P, two-pore-domain K+ channels; KATP, channels, inward-rectifying ATP-sensitive K+

channels; Kv channels, voltage-gated K+ _{channels; SK channel, small conductance Ca}2+_{-activated K}+ _channels;.