A flagellar K ⁺ -dependent Na ⁺ /Ca ²⁺ exchanger keeps Ca ²⁺ low in sea urchin spermatozoa

Abstract

The metabolism, flagellar beating, and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. As is true of most cells, swimming sperm maintain intracellular Ca 2؉ concentrations at submicromolar levels. Here we describe a K ؉ -dependent Na ؉ ͞Ca 2؉ exchanger (suNCKX) from sea urchin sperm. The suNCKX is phylogenetically related to other NCKXs, which use high relative intracellular K ؉ , and high relative extracellular Na ؉ , to couple the efflux of 1 Ca 2؉ and 1 K ؉ to the influx of 4 Na ؉ . The 652-aa suNCKX shares structural topology with other NCKX proteins, and has two protein kinase A sites and a His-rich region in its cytoplasmic loop. The suNCKX is encoded by a single gene, which is highly expressed in testes. The suNCKX activity of whole sperm shows Na ؉ and K ؉ dependence, and like other NCKXs can run in reverse exchange mode. An inhibitor blocks the suNCKX activity and sperm motility. suNCKX localizes to the plasma membrane over the sperm flagellum. The suNCKX may play a major role in keeping Ca 2؉ low in swimming sperm.sperm motility ͉ ion exchangers ͉ fertilization ͉ flagellar ion exchange

Mechanism Regulating Ca2+-dependent Mechanosensory Behaviour in Sea Urchin Spermatozoa

“…The plasma membrane proteins contributing to Ca 2+ efflux that have been demonstrated in sea urchin sperm are the plasma membrane Ca 2+ -ATPases (suPMCA) and the K + -dependent Na + /Ca 2+ exchanger (suNCKX) (Su and Vacquier, 2002;Gunaratne et al, 2006). We examined the effects of a blocker of the suPMCA, 5(6)-carboxyeosin (CE) (Gunaratne et al, 2006), and a blocker of the suNCKX, KB-R7943 (Su and Vacquier, 2002;Shiba et al, 2006;Rodríguez and Darszon, 2003), on the avoiding response. It has been reported that the motility of the swimming sperm is largely inhibited by CE at 5 μM (Gunaratne et al, 2006).…”

“…Among candidate proteins that mediate Ca 2+ efflux from cells, plasma membrane Ca 2+ ATPases (suPMCA) and K + -dependent Na + /Ca 2+ exchangers (suNCKX) have been well characterized in sea urchin sperm (Su and Vacquier, 2002;Gunaratne, et al, 2006). Their importance for the normal swimming of sperm has been suggested, because their blockers 5(6)-carboxyeosin (CE) and KB-R7943 inhibit the (quiescence) with a cane-shape waveform (Q), a straight path with more symmetrical bending waves than those of CB (S), and a circular path after MS with more asymmetrical bending waves (CA) in the presence of 10 mM Ca 2+ in ASW.…”

“…Gadolinium, cobalt and verapamil were dissolved in water, and pimozide, CE and KB-R7943 were dissolved in dimethyl sulfoxide. When the sperm were suspended in ASW for observation of their swimming, each blocker was added to ASW to obtain final concentrations as follows: 10-40 μM Gd 3+ , 3 mM Co 2+ , 0.1 mM verapamil, 10 μM pimozide, 1-3 μM CE and 1-2 μM KB-R7943 (Wakabayashi et al, 2009;Gunaratne et al, 2006;Su and Vacquier, 2002). For the experiments of Gd 3+ , we used ASW without EDTA.…”

Niflumic acid disrupts marine spermatozoan chemotaxis without impairing the spatiotemporal detection of chemoattractant gradients

“…7A,ii,iii). This hyperpolarization could activate the NCKX that extrudes Ca 2+ from the cell, thus shaping the [Ca 2+ ] i increase into a transient fluctuation (Jayantha Gunaratne and Vacquier, 2007;Su and Vacquier, 2002;Nishigaki et al, 2004;Kashikar et al, 2012). This hyperpolarization could also open HCN channels, depolarizing the V m again, and leading to the opening of CaV channels and setting up a cycle of Ca 2+ influx and efflux through the concerted action of HCN, CaV, CaCC and/or CaKC.…”

“…Current models propose that the binding of speract to its receptor promotes the synthesis of cGMP that activate K + selective and cyclic nucleotide-gated channels (KCNG) leading to membrane potential (V m ) hyperpolarization Strünker et al, 2006;Bönigk et al, 2009). This V m change first induces a pH i increase (Nishigaki et al, 2001;Nishigaki et al, 2004), stimulates hyperpolarization-activated and cyclic nucleotide-gated channels (HCN) (Gauss et al, 1998), removes the inactivation of voltage-gated Ca 2+ channels (CaV) (Strünker et al, 2006;Granados-Gonzalez et al, 2005), and facilitates Ca 2+ extrusion by Na + /Ca 2+ exchangers (NCKX) (Jayantha Gunaratne and Vacquier, 2007;Su and Vacquier, 2002;Nishigaki et al, 2004). The opening of HCN and the influx of Na + contribute to V m depolarization, and concomitant increases in [Ca 2+ ] i and [Na + ] i further depolarize V m .…”