As mentioned above, the channels involved in the formation of oscillatory electrical activity in Beta cells are very diverse, the main ones being KATP channels regulated by the intracellular ADP/ATP ratio, voltage-dependent L, P/Q, R and N-type Ca channels, voltage-dependent Ca inward current and intracellular Ca inward current. BK channels triggered by the emptying of Ca stores, delayed rectifier K channels can be counted. In this title, the general features of these channels and other minor channels will be mentioned.
3.1 ATP regulated K channels (KATP Channels)
KATP channels in beta cells are channels belonging to the family of ATP-regulated K channels in cardiac muscle, smooth muscle, and skeletal muscle, and they are the channels that make the greatest contribution to the negative resting membrane potential in beta cells [25, 26]. It provides an ionic gradient with [K+]o on average 5mM and [K+]i on average 150 mM (net 140–145 mM) while operating, and the resting Beta cell membrane reaches an electrical value of -70mV, which is very close to the K equilibrium value [27–29]. One of the greatest features of these channels is that they contain Na+ and Mg+ 2 binding sites on their inner surfaces. These cations prevent K outflow from the channels at certain concentrations (34 mmol, Na+; 46mmol Mg+ 2) [30]. While the single channel permeability of the channels is highest at 140mM external K concentrations, it shows saturation with [K]o increase and reaches 50% saturation at 220mM external K concentration [30].
When its kinetics are examined, it is noteworthy that it opens in groups and explosively, and there is an interval in which it remains closed after each explosion. The bursting opening voltages of each group of channels and the duration of the intervals during which they remain closed differ from each other, and the opening voltages vary between − 60 and − 90mV on average, and the duration of openness in each burst varies between 0.5-2ms [31]. The dominant effect of ATP is to decrease the number of channels opened in each burst, to make the bursts last shorter and to increase the duration of the intervals [32].
It is noteworthy that besides the inhibition on KATP channels, ATP also provides reusability of channels by binding with magnesium [33, 34].
3.1.a Inhibition mechanism of ATP on KATP
The inhibition of ATP on KATP channels depends on its intracellular concentration, and it has been shown that ATP in the extracellular environment does not cause inhibition on the channels. Although ATP is a highly negatively charged molecule and its increased concentration in the cell makes the membrane potential negative, the inhibitory effect of ATP on these K channels is independent of membrane potentials [35]. In studies, even at different membrane potentials, 50% inhibition was observed when intracellular ATP reached 50 mM level and full inhibitory effect was observed when it reached millimole level, and therefore it was suggested that ATP has a specific binding site on K channels [36–38].
When we look at the structure of KATP channels in general, it is observed that they are in octamer structure and consist of 4 alpha (Kir6.2) and 4 Beta (Sulfonylurea receptor [SUR]) subunits [39, 40]. Alpha subunits are common in all tissues and participate in pore formation [41]. Beta subunits are considered as the regions where ATP binds to create inhibition and have regulatory functions. Three different forms of these beta subunits have been identified: SUR1, SUR2A and SUR2B. Unlike alpha subunits, these forms differ according to the tissues in which the channels are located [42, 43] (Fig. 2).
Figure 2. The structure of KATP channels in beta cells.
While Kir6.2s, which take place as alpha subunits in KATP channels, are responsible for forming pores, beta subunits are composed of Sulfonylurea receptors (SUR) types. In the figure, it is observed that KATP in pancreatic beta cells contains especially SUR1 and the nucleotide binding domain (NBD) structures of SUR1 (Adapted from: [44]).
By binding of ATP to Beta subunits, it creates a closure in Kir6.2, which is in the neighborhood, and creates depolarization in the cell. In other words, it is the alpha subunit that creates the actual depolarization. Such that, as a result of Kir6.2 mutation, persistent hyperinsulinemic hypoglycemia picture occurs in infants due to uncontrolled beta cell depolarization and insulin secretion [45] (Fig. 3).
Figure 3. Modulation of KATP Channels.
A. ATP binding to SUR1 subunit and Kir6.2 inhibition as a result of increased glucose levels. B. Activation of Kir6.2 and increase in [K+]o current in the absence of glucose. C. Decreased permeability of the duct and uncontrolled insulin secretion as a result of the mutation observed in infantile persistent hyperinsulinemic hypoglycemia (Adapted from: [46]).
Not all of the complexes formed by beta subinides reach the cell membrane, and some of them are linked to the smooth endoplasmic reticulum and are regulated by Ca+ 2 signaling [47]. Although this bond is more common in the SUR2A subtype, which is located on smooth muscle and cardiac muscle and regulates contraction, it has also been observed in the SUR1 subtype located in pancreatic beta cells [48, 49]. Although ATP creates inhibition by binding to alpha subunits, beta subunits modulate the permeability of channels by modulating ATP sensitivity [50–52]. Particularly, the subtype SUR1, located in beta cells, shows greater sensitivity to sulfonylurea drugs [53], Mg-ADP complex [54] and metabolic stress [55].
Although the exact mechanism by which ATP inhibits KATP channels is still unclear, phosphorylation-mediated modulation has been shown to play a role in this mechanism. Among the studies on this subject, it was shown for the first time in 1989 that phosphorylation of KATP channels prevents K+ outflow [56]. In later studies, it was found that KATP channels were activated by phosphorylating in vascular smooth muscle by means of protein kinase A (PKA) [57, 58], and stimulation of D1 receptor of dopamine in renal artery vascular smooth muscle was found. It has been shown that it activates KATP channels by stimulating protein kinase A and exerts a vasodilator effect due to hyperpolarization [59]. However, Hatakeyama et al. suggested that PKC-dependent phosphorylation causes vasoconstriction by creating KATP inhibition in vascular smooth muscle cells [60]. This difference in the effects of PKA and PKC suggests that the two enzymes provide phosphorylation from different sites. Lin et al. suggested that the effects of protein kinases are still observed in the SUR1 subunit (the beta subunit specific to pancreatic beta cells) knockout cells, therefore the alpha subunit (Kir6.2) phosphorylated for modulation purposes [61]. Indeed, 2 phosphorylation sites have been identified on Kir6.2 (Serine 372 and Threonine 224), and it has been shown that the main site to be phosphorylated in channel activation is T224 [61]. This suggests that the channels may be inhibited by phosphorylation of ATP via S372 via PKC in KATP channels.
On the other hand, although ATP is the key molecule in KATP channel activation, it has been shown that ATP cannot adequately induce channel inhibition in the presence of intracellular ADP and GDP [62–64]. Since there is a decrease in the amount of ADP with glucose metabolism, many studies have investigated the effects of ADP as well as ATP on KATP channels and showed that the effect of the change in ADP/ATP ratio on the channels is stronger than the effect of ATP alone [65, 66]. In the study of Kakei et al., it was shown that KATP activity was not inhibited even if [ATP]i was increased to millimolar concentrations, provided that the ADP/ATP ratio similar to intact beta cells was maintained [62]. How ADP inhibits ATP-induced inhibition is still unclear. Some studies have suggested that ADP acts as a weak agonist on KATP and competes with ATP, preventing its binding, thereby weakening its effect [63, 67]. However, the need for Mg+ 2, a positive ion in the cell, while performing this task, and the inability to inhibit ATP even at high levels in the absence of Mg+ 2, suggested that the areas where ATP and ADP bind might be different [68].
3.1.b Activation and repolarization of KATP channels
Various studies suggest that Mg-ATP or its complex reactivates the channel, while increasing ADP/ATP ratios in Mg deficiency are not effective in removing the inhibition of the channel [69, 70]. In parallel, diazoxide-derived drugs, which increase the efficiency of KATP channels, hyperpolarize beta cells and suppress insulin secretion, cannot have the expected effect in patients with insulinoma. The effect of diazoxide treatment is parallel to the intracellular Mg-ATP concentration [71, 72]. On the other hand, it has been shown that the ADP-Mg complex rather than the Mg-ATP complex has a more potent effect on the activation of the channel, which may explain the reason for the disappearance of the inhibition on the channel in the presence of Mg+ 2 as a result of the increase in the ADP/ATP ratio [73] (Fig. 4).
Figure 4. Signals responsible for the reactivation of Kir6.2s.
The increased intracellular Mg-ATP complex reopens Kir 6.2 via SUR1s, providing [K+]o current and ending the depolarization that provides insulin release. Diazoxide also acts like Mg-ATP.
Many phosphatase enzymes work in an intracellular Ca+ 2 dependent manner or use Mg as a cofactor. However, since most of the studies on KATP buffer intracellular Ca+ 2, it can be said that calcium does not have much of a role at this point. In this case, it comes to mind that Mg+ 2 may be involved in the dephosphorylation of KATP. Indeed, it is noteworthy that the activation of KATP in beta cells by phosphatases is increased, but this effect cannot be observed with buffering of intracellular Mg+ 2 [74, 75].
3.2 Delayed rectifier K channels
Delayed rectifier K channels (KDR) were first demonstrated in pancreatic beta cells in rat insulinoma serial cells (RINm5f) in 1986 [76], and were found in human pancreatic beta cells in 1990 [77]. Potassium current in KDR channels is observed as a slow outflow that starts during depolarization and continues throughout repolarization. In studies, its activation begins at -30mV under physiological conditions and increases sigmoidally during depolarization [76, 78]. When voltage dependent states are examined in-vitro, it shows half-maximal activation at -20mV and an activation time of 2ms at 0mV [79]. Afterwards, it slows down after a slope factor of 2-8mV and provides a -50mV return in the membrane potential with an activation time of 30ms. At high extracellular K+ concentrations, activation potentials decrease down to -50mV, but activation times decrease 2–3 times [79]. This situation, by decreasing the required K+ outflow from beta cells, causes the prolongation of the plateau observed in the membrane and shortens the intervals, thus triggering insulin secretion. This provides insight into how hyperkalemia increases insulin release.
On the other hand, in-vivo voltage values in humans and rats differ compared to in-vitro. Simth et al. showed that the opening of KDR channels is regulated by divalent cations, and the opening potential of the channels shifts to approximately 10mV positive at an increase of 10mM Ca+ 2 [79]. Therefore, physiologically, in the presence of 5mM extracellular ionized calcium, the channels showed half-maximal activity at a value of -2mV [77]. In chronic hypercalcemia states, this may affect the electrical potential of the cells by causing an increase in intracellular K+ in beta cells, and may disrupt the balance of insulin secretion by making it difficult to transition from the plateau phase to intervals. Similarly, Bacerra-Thomas et al. stated that chronic hypercalcemia may pose a risk for type-2 diabetes by impairing insulin secretion [80].
3.2.a Inactivation of delayed rectifier K channels
KDR channels are slowly inactivated during depolarization, and half-maximal inactivation voltages are stated as + 20mV in pancreatic beta cells [76]. On the other hand, some pharmacological agents have been shown to cause inactivation of beta cell KDR channels. One of them, quinine, has been observed to block the current in the channels by binding to more than one region on a single channel at 4µM levels. As a result, an approximately 80% shortening of the channels' open state times and a 25% prolongation of their closure times occurs [81]. Another KDR blocker is to frequently lose weight and trigger insulin release. Although Forskolin is known as an adenylcyclase stimulator and it is stated that it produces insulin secretion with an increase in cAMP, it also increases insulin secretion by blocking the current in KDR channels [82]. On the other hand, no effect of sulfonylureas on KDR channels was observed [83].
3.3 Ca-dependent K channels (BK channels)
Big current Ca and voltage-dependent K channels (BK) in beta cells were first shown by patch-clamp studies in 1984 [84]. The properties of Ca-dependent K channels in beta cells have been demonstrated by various studies, some of which are as follows: decrease in Ca+ 2 concentrations in the extracellular environment has been shown to cause a decrease in the amplitude of [K]o current [85]. In single-cell recordings taken simultaneously, it has also been observed that the amplitude of [K]o currents in Beta cells decreases when Ca+ 2 entry into the cell is reduced by the use of Ca+ 2 antagonists [86] and high [K]o current was recorded while simultaneously [Ca]i currents were recorded [86]. In in-vitro studies, it has been shown that when Ca+ 2 is not buffered, BK channels are responsible for 60% of the total K outflow current in Beta cells, while the rate decreases to 20% when a physiological Ca level is buffered [87]. In parallel with the increase in intracellular Ca+ 2 concentration, the increase in BK channel activity and the increase in K+ outflow bring the membrane closer to a more negative membrane potential. Due to these properties, it can be thought that BK channels are responsible for the transition to the plateau phase from the explosive rise of the action potential in pancreatic beta cells, which is accompanied by Ca+ 2 elevation.
BK channels are not only dependent on the Ca+ 2 level, but also operate in a voltage-dependent manner. Although the minimum Ca+ 2 values required for the activation of the channels are around 4mmol/l, these values are valid when the membrane potential is close to + 20mV. At lower membrane potentials, higher concentrations of calcium are required for the activation of the channels. At a membrane potential of -50 to -20mV, which is the action potential treshold voltage range of pancreatic beta cells, the required Ca concentration has been shown to be 10-22mM, and the required Ca+ 2 concentration has been shown to be 30mM when the beta cell is at resting membrane potential [88]. BK channels do not contribute to resting K permeability, as human pancreatic Beta cells can never have such a high intracellular Ca+ 2 concentration under physiological conditions at rest. In humans, beta cell BK channels begin to activate at an average of 0mV during depolarization.
Activation of BK channels is also dependent on intracellular pH. While BK channel activation is maximum at pHi 7.6, channel activation stops at pHi 6.8 [84]. Although it triggers the outflow of intracellular buffers during the decrease in pHe and increases the Ca+ 2 entry into the cell and the increase in intracellular Ca+ 2 compensates for the decrease in BK activity [89], this situation loses its validity in chronic acidosis and decreases the [K]o current. Thus, electrical potential becomes positive and may disrupt the regulation of insulin secretion. There are studies showing that chronic acidosis creates insulin resistance by disrupting insulin regulation, and the most recent of them was 2021, and it would not be wrong to explain one of the underlying mechanisms in this resistance development as BK inhibition [90].
3.3.a Modulation of BK Channels
BK channels in beta cells have complex kinetics with two open and three closed states. Two of these three off states are observed during burst depolarization, and one is observed during intervals [81]. The opening and closing times of the channels are regulated by the above-mentioned mechanisms and intracellular Ca+ 2 levels [88]. The functions of BK channels are also regulated by intracellular glucose concentrations. The increase in intracellular glucose metabolism inhibits BK channels and contributes to the positivity of the membrane potential in the cell and insulin release [91].
Small-conductance calcium-activated K+ channels (SK) have also been demonstrated in pancreatic beta cells as well, but unlike BK channels, their mechanism of contribution to insulin regulation has not yet been fully elucidated [92].