The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa1

Abstract

Volume regulation is a necessary task for spermatozoa as the osmolarity of female tract fluids is lower than that in the epididymis and because the disruption of it in transgenic mice results in infertility. As the specific mechanisms behind this phenomenon are unknown, spermatozoa from mice were screened for sensitivities to inhibitors known to affect specific channels involved in volume regulation of somatic cells. Spermatozoa from the cauda epididymidis were exposed to physiological hypotonic conditions with and without inhibitor. Flow cytometric forward scatter measurements were taken to indicate relative sperm size at 5 and 75 min of incubation. The presence of quinine (0.8 mM), cadmium (0.2 mM), flecainide (100 microM), 4-aminopyridine (4 mM), barium (1 mM), clofilium (10 microM), and phrixotoxin (100 nM) for 75 min resulted in significantly higher forward scatter values than sperm incubated in medium without an inhibitor. These results imply that channels potentially involved in volume regulation of murine spermatozoa include the voltage-dependent Kv1.4 (also known as KCNA1), Kv1.5 (KCNA5), Kv4.1 (KCND1), Kv4.2 (KCND2), Kv4.3 (KCND3), mink (KCNE1), and acid-sensitive TASK2 (KCNK5) and TASK3 (KCNK9). Western blots confirmed the presence of Kv1.5 and TASK2 proteins in sperm plasma membranes at similar (Kv1.5) or higher (TASK2) molecular weight than in somatic cells. Incubation in a different pH did not reveal acid sensitivity of volume regulation. Volume regulation of spermatozoa may involve novel voltage-gated and pH-sensitive potassium channels, which could be valuable targets for the development of a posttesticular male contraceptive.

Expression of two-pore domain potassium channels in nonhuman primate sperm

“…Sperm samples demonstrated specific K 2P protein bands at approximately 100 kd, consistent with the expected molecular weights (92-117 kd) of the various K 2P channels reported previously under similar conditions (11,21,22) (Fig. 1A-C).…”

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

“…TASK-2 is sensitive to clofilium (35), independent of intracellular Ca 2ϩ (35,41), and highly sensitive to external pH (21). Besides being the swelling-activated K ϩ channel in EATC, TASK-2 has also been found to be involved in volume regulation in kidney cells (4) and in murine spermatozoa (1). Moreover, TASK-2 has been associated with apoptotic volume decrease in EATC cells following cisplatin exposure (40).…”

KCNK5 is Functionally Down-Regulated Upon Long-Term Hypotonicity in Ehrlich Ascites Tumor Cells

“…In EAT cells and Ehrlich lettré ascites (ELA) cells the volume sensitive channels are the two-pore domain K + channel KCNK5 (also known as TASK-2 - TWIK-related Acid-Sensitive K + channel 2 or K 2P 5.1) [5,6,7], and the volume regulated anion channel (VRAC, I Cl,vol ) [8]. KCNK5 has, besides in Ehrlich cells [5,6,7], also been shown to be involved in RVD in other cell types including mouse proximal tubules [9], T lymphocytes [10,11], murine spermatozoa [12] and retinal glial cells [13]. It has previously been shown that the rate limiting factor in RVD in EAT cells is the volume sensitive K + efflux and thus the efflux through the KCNK5 channel [14], making an altered RVD response very likely to be due to changes related to this channel.…”