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Phill Stansfeld publications

PNAS aug21 75x100An outer pore gate controls the pharmacology of the TMEM16A channel

Ria L. Dinsdale, Tanadet Pipatpolkai, Emilio Agostinelli, Angela J. Russell, Phillip J. Stansfeld and Paolo Tammar

TMEM16A Ca2+-activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca2+ was omitted. However, when physiological Ca2+ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca2+ is mandatory for the channel response to an extracellular modulator.

PNAS. August 2021

Nature CommunicationsDeciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

Jakob M Silberberg, Robin A Corey, Lisa Hielkema, Charlotte Stock , Phillip J Stansfeld, Cristina Paulino, Inga Hänelt

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

Nature Communications. August 2021

Mon 27 Sep 2021, 08:22