Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation

Abstract

Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

Methamphetamine Regulation of Firing Activity of Dopamine Neurons

“…The BK-␣ subunit is the pore-forming unit of the BK channel (Knaus et al, 1994), and it has been shown that there is a direct relationship between BK channel regulation of neuronal excitability and the level of BK-␣ subunits at the surface membrane (Shruti et al, 2012;Chen et al, 2013;Cox et al, 2014). Therefore, we examined the possibility that METH-mediated suppression of neuronal activity is due to METH-induced reduction of the BK-␣ subunit level at the plasma membrane.…”

“…For example, activation of BK channels has been shown to enhance the excitability of adrenal chromaffin cells (Solaro et al, 1995;Lovell and McCobb, 2001), CA1 hippocampal pyramidal cells (Gu et al, 2007), and parasympathetic cardiac motoneurons in the nucleus ambiguus . Similarly, enhancement of BKchannel currents has been linked to neuronal hyperexcitability and epilepsy in cortical neurons (Brenner et al, 2005;Lorenz et al, 2007;Shruti et al, 2008;Wang et al, 2009).…”

“…Our laboratory and others have shown that acutely applied amphetamines (1-3 min) increases the firing activity of dopamine neurons (Ingram et al, 2002;Saha et al, 2014). Dopamine transporter activity and different ionic currents involved in action potentials (APs), such as L-type calcium current (Shibasaki et al, 2010), outward potassium current (Lin et al, 2010), and inwardly rectifying potassium current (Sharpe et al, 2015), are thought to be directly or indirectly associated with METH regulation of electrical properties of neurons, albeit with a mechanism that is less understood in dopamine neurons.…”

“…Large-conductance Ca 2ϩ -activated potassium channels (KCa1.1, BK, MaxiK) play important roles in controlling neuronal excitability (Shao et al, 1999;Faber and Sah, 2002;Lin et al, 2014). BK channels have been shown to mediate a link between intracellular signaling pathways and electrical signals in the neurons through a feedback regulation countering increases in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) and membrane depolarization (Vergara et al, 1998;Latorre and Brauchi, 2006;Salkoff et al, 2006).…”

“…METH has been shown to increase protein levels of the L-type calcium channel Ca v 1.2 and Ca v 1.3 in the frontal cortex and limbic forebrain neurons (Shibasaki et al, 2010), leading to increased intracellular Ca 2ϩ levels (Uramura et al, 2000). Moreover, METH-induced inward Na ϩ current via the dopamine transporter has been shown to depolarize dopamine neurons (Fischer and Cho, 1979;Stephans and Yamamoto, 1995;Saha et al, 2014).…”