Molecular Basis of Slo Channel(S) Function in Sperm Revealed by Human Genetics

Abstract

Flagella potassium channel of sperm (KSper) is required for successful fertilization. In mice the dominant K+ conductance is via KCNU1 or Slo3, a pH regulated K+ channel. In humans KSper is activated by Ca2+, not pH, and is inhibited by scorpion toxin. Based on these unique properties, it was suggest that Slo1 (KCNMA1) underlies the molecular identity of human KSper. Recent conflicting work indicated that human KSper maybe a Ca2+ sensitive Slo3 variant. Due to the lack of a Ca2+ sensing domain in the structure of Slo3, it seems unlikely that it would be activated by Ca2+. Therefore to address this conundrum, we characterized a KSper defective patient and present here the molecular composition of this channel complex. Infertile patients attending Ninewells Hospital, Dundee, were screened for KSper current (IK+) by using sperm patch-clamp method. One out of 13 patients showed a complete loss of IK+ and was insensitive to changes in intracellular Ca2+. MiSeq genome analysis showed no mutations in KCNMA1 but a single nucleotide polymorphism (SNP) at position 768 (W768R) in KCNU1 was present. Electrophysiological assessment of heterologously expressed mutated KCNU1 in CHO cells cloned from human spermatozoa showed no loss IK+. Further analysis of patient genome showed additional mutation in auxiliary subunits which were not present in fertile donors. Co-expression of mutated subunits in CHO cells showed a complete loss of IK+ identical to patient. Interestingly, we were unable to show Ca2+ activated IK+ in CHO cells with wild type KCNU1. This suggests human KSper may be more complex than initially thought and requires an additional Ca2+ sensitive module for channel activation.