recording-electrical-currents-across-plasma-membrane-mammalian-sperm

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

University of California, San Francisco

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

Rediscovering sperm ion channels with the patch-clamp technique.

Adaptive thermogenesis regulates core body temperature, controls fat deposition, and contributes strongly to the overall energy balance. This process occurs in brown fat and requires uncoupling protein 1 (UCP1), an integral protein of the inner mitochondrial membrane. Classic biochemical studies revealed the general principle of adaptive thermogenesis: in the presence of long-chain fatty acids (FA), UCP1 increases the permeability of the inner mitochondrial membrane for H, which makes brown fat mitochondria produce heat rather than ATP. However, the exact mechanism by which UCP1 increases the membrane H conductance in a FA-dependent manner has remained a fundamental unresolved question. Recently, the patch-clamp technique was successfully applied to the inner mitochondrial membrane of brown fat to directly characterize the H currents carried by UCP1. Based on the patch-clamp data, a new model of UCP1 operation was proposed. In brief, FA anions are transport substrates of UCP1, and UCP1 operates as an unusual FA anion/H symporter. Interestingly, in contrast to short-chain FA anions, long-chain FA anions cannot easily dissociate from UCP1 due to strong hydrophobic interactions established by their carbon tails, and a single long-chain FA participates in many H transport cycles. Therefore, in the presence of long-chain FA, endogenous activators of brown fat thermogenesis, UCP1 effectively operates as an H uniport. In addition to their transport function, long-chain FA competitively remove tonic inhibition of UCP1 by cytosolic purine nucleotides, thus enabling activation of the thermogenic H leak through UCP1 under physiological conditions.

UCP1: A transporter for H and fatty acid anions.

Cold and other environmental factors induce "browning" of white fat depots-development of beige adipocytes with morphological and functional resemblance to brown fat. Similar to brown fat, beige adipocytes are assumed to express mitochondrial uncoupling protein 1 (UCP1) and are thermogenic due to the UCP1-mediated H leak across the inner mitochondrial membrane. However, this assumption has never been tested directly. Herein we patch clamped the inner mitochondrial membrane of beige and brown fat to provide a direct comparison of their thermogenic H leak (I). All inguinal beige adipocytes had robust UCP1-dependent I comparable to brown fat, but it was about three times less sensitive to purine nucleotide inhibition. Strikingly, only ∼15% of epididymal beige adipocytes had I, while in the rest UCP1-dependent I was undetectable. Despite the absence of UCP1 in the majority of epididymal beige adipocytes, these cells employ prominent creatine cycling as a UCP1-independent thermogenic mechanism.

Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling.

Uncoupling protein 1 (UCP1) is an integral protein of the inner mitochondrial membrane (IMM) that is expressed specifically in brown and beige fat depots. UCP1 is responsible for the production of heat to control core body temperature, the regulation of fat metabolism, and the energy balance. As an uncoupling protein, UCP1 transports H across the IMM in presence of long-chain fatty acids (FA), which makes brown fat mitochondria produce heat at the expense of ATP. However, the exact mechanism of UCP1 action has remained difficult to elucidate, because direct methods for studying currents generated by UCP1 were unavailable. Recently, the patch-clamp technique was successfully applied to brown and beige fat mitochondria to directly study H currents across the IMM and characterize UCP1 function. A new model of the UCP1 mechanism was proposed based on the patch-clamp analysis. In this model, both FA anions (FA) and H are transport substrates of UCP1, and UCP1 operates as a non-canonical FA/H symporter. Here, we summarize recent findings obtained with the patch-clamp technique that describe how UCP1 can transport not only H but also FA.

The Mechanism FA-Dependent H Transport by UCP1.

Mitochondria accumulate significant amounts of calcium when cytosolic calcium is elevated above the resting levels of 50-100 nM during signaling events. This calcium uptake is primarily mediated by a macromolecular protein assembly called mitochondrial calcium uniporter (MCU) that resides in the mitochondrial inner membrane. In 2004, we applied patch-clamp technique for the first time to record calcium currents from the mitochondrial inner membrane and proved unequivocally that MCU is a highly selective calcium channel. This chapter describes how patch-clamp technique can be applied to record the Ca uniporter currents from the mitochondrial inner membrane, isolation of mitochondria from the heart tissue, and preparation of mitoplast using French Press. We also discuss advantages of patch-clamp technique as compared to other methods of determining mitochondrial uniporter activity and important considerations in applying patch-clamp technique to such a small subcellular organelle. With small variations in the bath and pipette solution composition, the same methodology can be applied to study any other currents (e.g., H or Cl) from the mitochondrial inner membrane.

Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter.

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H transport. The AAC-mediated H current requires free fatty acids and resembles the H leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H leak, but does not completely inhibit it. This suggests that the H leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

Article	Year
Rediscovering sperm ion channels with the patch-clamp technique. Molecular human reproduction\| PubMed ID: 21642646	2011
UCP1: A transporter for H and fatty acid anions. Biochimie\| PubMed ID: 27984203	2017
Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling. Cell metabolism\| PubMed ID: 28380374	2017
The Mechanism FA-Dependent H Transport by UCP1. Handbook of experimental pharmacology\| PubMed ID: 29797089	2019
Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30674018	2019
H transport is an integral function of the mitochondrial ADP/ATP carrier. Nature\| PubMed ID: 31341297	2019

Yuriy Kirichok

Biography

JoVE Journal Publications

Other Publications