Phosphatidylinositol 3-Phosphate Indirectly Activates KCa3.1 via 14 Amino Acids in the Carboxy Terminus of KCa3.1

Abstract

KCa3.1 is an intermediate conductance Ca2؉ -activated K ؉ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca 2؉ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca 2؉ gating of KCa3.1. INTRODUCTIONKCa3.1 subunits (also known as IKCa and KCNN4) are components of an intermediate conductance Ca 2ϩ -activated K ϩ channel that is found predominantly in peripheral tissues such as blood cells, epithelia, and smooth muscle cells where it functions to couple alterations in cytosolic Ca 2ϩ to K ϩ flux Jensen et al., 2001;Wulff et al., 2003). KCa3.1 channels thereby play an important physiological role to set the membrane potential at negative values close to the K ϩ equilibrium potential Jensen et al., 2001;Wulff et al., 2003;Stocker, 2004). This effect on membrane potential can have diverse physiological responses in a variety of cell types, including water movement and volume regulation in red blood cells, mitogen activation of T-lymphocytes, Cl Ϫ secretion of exocrine epithelial cells, and control of proliferation by a variety of cells such as Tand B-lymphocytes, vascular smooth muscle cells, keratinocytes, and some cancer cell lines (Khanna et al., 1999;Ghanshani et al., 2000;Fanger et al., 2001;Koegel and Alzheimer, 2001;Kohler et al., 2003;Maher and Kuchel, 2003; OuadidAhidouch et al., 2004;Wulff et al., 2004). Based on these activities, pharmacological modulation of KCa3.1 channels has been proposed to treat proliferative diseases such as restenosis after angioplasty and cancer, transplant rejection as well as secretory diarrheas, sickle cell anemia, and cystic fibrosis (Rufo et al., 1997;Jensen et al., 2001;Maher and Kuchel, 2003;Wulff et al., 2003).Over the past several years, the mechanism of activation of KCa3.1 and the three related small conductance Ca 2ϩ -activated K ϩ channels, KCa2.1, KCa2.2, and KCa2.3 channels (also known as SK1, SK2, and SK3), has been determined. These channels are all structurally similar and possess...

T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase

“…For instance, mice deficient for mitsugumin-29 display similar ultrastructural abnormalities, but only minor defects in muscle strength (37). Myotubularin activity on PI(3)P and/or PI(3,5)P 2 could also play a direct role on channel activation, as previously reported for a homologous protein, MTMR6, which regulates the activity of the Ca 2ϩ -activated K ϩ channel KCa3.1 via PI(3)P (38,39). Other mutant mice with deletions in genes involved in triad formation and/or calcium homeostasis, such as junctophilin 1 and triadin, also contain muscles with abnormally oriented T-tubules and alterations in Ca 2ϩ transients and/or E-C coupling (40) (41).…”

Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis

“…Together, these findings suggest that macropinocytosis is controlled by the sequential breakdown of PI(3,4,5)P 3 → PI(3,4)P 2 → PI(3)P → PI in the PM. Furthermore, we showed that KCa3.1, a Ca 2+ -activated K + channel that is activated by a PI(3)P (26)(27)(28), is essential for macropinocytosis.…”

Specificity of the Myotubularin Family of Phosphatidylinositol-3-phosphatase Is Determined by the PH/GRAM Domain

“…We recently found that the calcium-activated potassium channel KCa3.1 requires PI(3)P for activity and is specifically inhibited by MTMR6 (21)(22)(23). These studies demonstrated that interaction between the CC domain of MTMR6 with the CC domain in the carboxyl terminus of KCa3.1 is critical for the inhibition of KCa3.1 and proposed that interaction between the CC domains on KCa3.1 and MTMR6 functions to localize MTMR6 at the PM where it then dephosphorylate PI(3)P leading to KCa3.1 inhibition.…”