The Phosphatidylinositol 3-Phosphate Phosphatase Myotubularin- Related Protein 6 (MTMR6) Is a Negative Regulator of the Ca²⁺-Activated K⁺ Channel K_Ca3.1

Abstract

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3 position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 . MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca 2؉ -activated K channel, K Ca 3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K Ca 3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K Ca 3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K Ca 3.1, indicating that MTM CC domains are sufficient to confer target specificity. K Ca 3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K Ca 3.1 by MTMR6 overexpression. These data, when taken together, indicate that K Ca 3.1 is regulated by PI(3)P and that MTMR6 inhibits K Ca 3.1 by dephosphorylating the 3 position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K Ca 3.1. K Ca 3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K Ca 3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.Myotubularins (MTM) are a large family of evolutionarily conserved lipid phosphatases (PT) that specifically dephosphorylate the 3Ј position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P 2 (28, 39). Fourteen MTMs in mammalian cells have been identified, and they can be divided into six subgroups based on sequence alignment and phylogenetic comparison. Members of one of these subgroups lack phosphatase activity due to a mutation in a critical residue within the phosphatase domain. MTM1, the founding member of this gene family, is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) syndrome type 4B (3, 30, 37). In addition to containing a phosphatase domain, most MTMs are composed of a GRAM domain which may mediate association of MTMs with membranes, a Rac-induced localization domain which mediates the association with Rac-induced membrane ruffles, and a C-terminal coiled coil (CC) domain (10,28,29,39). Recent data have indicated that the MTM CC domains mediate specific heterodimerization between a MTM (PT ac...

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).…”

A phosphoinositide conversion mechanism for exit from endosomes

“…Thus, Ca 2+ release from the sarcolemma is initiated by excitation. Additionally, local PI homeostasis of PI(3)P and PI(3,5)P 2 , controlled by myotubularins, was shown to regulate ion channel activity (Srivastava, Li et al 2005;Shen, Yu et al 2009). Defective membrane organization and channel activity might have additive effects on muscle dysfunction, reflected by the severe form of myotubular myopathy.…”

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.…”