Inhibition of CFTR Cl ⁻ channel function caused by enzymatic hydrolysis of sphingomyelin

Abstract

Numerous mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR, a Cl − channel) disrupt salt and fluid transport and lead to the formation of thick mucus in patients' airways. Obstruction by mucus predisposes CF patients to chronic infections and inflammation, which become gradually harder to control and eventually fatal. Aggressive antibiotic therapy and supportive measures have dramatically lengthened CF patients' lives. Here, we report that sphingomyelinases (SMase) from human respiratory pathogens strongly inhibit CFTR function. The hydrolysis of sphingomyelin by SMase makes it more difficult to activate CFTR by phosphorylation of its regulatory domain. By inhibiting CFTR currents, SMase-producing respiratory tract bacteria may not only aggravate pulmonary infection in some CF patients but may also elicit a condition, analogous to CFTR deficiency, in non-CF patients suffering from bacterial lung infection.

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

“…PC-PLC from B. cereus was purchased from Sigma-Aldrich. To produce recombinant SMases C and D 30 , E. coli BL21 (DE3) cells were transformed with the respective cDNAs cloned into pET30 vector, grown in LB medium to ~0.6 OD at 600 nm, and induced with 1 mM IPTG for 2 hours. The bacteria were harvested, resuspended, and sonicated.…”

Sphingomyelinase D inhibits store-operated Ca2+entry in T lymphocytes by suppressing ORAI current

“…The SMase D effect on Kv channels persists even 24 h after treatment (Xu et al, 2008). Exposure to exogenous sphingomyelin or C1P fails to reproduce these functional effects in either channel type (Ramu et al, 2006(Ramu et al, , 2007. These findings suggest that the channel-impacting lipid molecules remain tightly bound to channel proteins, as they demonstrably do in the KcsA channel.…”

“…Hydrolysis of sphingomyelin by SMase D has been shown to suppress the CFTR Cl  channel and to lower the activation voltages of voltage-gated ion channels (Ramu et al, 2006(Ramu et al, , 2007Milescu et al, 2009;Combs et al, 2013). The SMase D effect on Kv channels persists even 24 h after treatment (Xu et al, 2008).…”

“…SMase C, were generated as described previously (Ramu et al, 2007) (the SMase D cDNA was provided by S. Billington, University of Arizona, Tucson, AZ). In all experiments, SMase D and SMase C were applied at 0.3 µg/ml, and the inactive form of SMase D was applied at 3 µg/ml.…”

Xenopus oocyte prophase I meiotic arrest is released independently from a decrease in cAMP levels or PKA activity

“…2E), as previously reported (Button et al, 2001). To minimize this, we pre-treated oocytes with 3-isobutyl-1-methylxanthine (IBMX) (Ramu et al, 2007), a phosphodiesterase inhibitor, to raise cAMP and activate PKA (Sadler and Maller, 1987;. IBMX pre-treatment significantly (P=0.0002) increased I CFTR , and limited its rundown to maintain a significant current for up to 25 min (Fig.…”