Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed C␣ 2 or Cs. We generated mice that lack C␣2 to determine its importance in the events downstream of cAMP production. Male C␣ 2 null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, C␣ 2 has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that C␣ 2 is required for bicarbonate to speed the flagellar beat and facilitate Ca 2؉ entry channels. In addition, C␣2 is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, C␣ 2 null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility. T he terminally differentiated, transcriptionally dormant, and translationally inactive posttesticular spermatozoan has a limited ability to respond to environmental cues encountered as it progresses through the male and female reproductive tracts. During this passage, the bicarbonate anion present in the reproductive fluids (1, 2) has an unexpectedly prominent role in promoting several of the events (3-5), collectively called capacitation, which transform sperm to readiness for fertilization.Much evidence indicates that bicarbonate directly increases production of cAMP by atypical sperm adenylyl cyclase (sAC) (6, 7), and recent work finds that sAC is required for male fertility and normal sperm motility (8). Although cAMP might open ion channels or activate guanine nucleotide exchange factors, most evidence places cAMP-dependent protein kinase (PKA) as the major downstream effector of cAMP signals in sperm. However, only a small number of sperm proteins have been identified as phospho-substrates of PKA (9, 10). We now apply phenotypic analysis of loss-of-function mutants to examine downstream effects of the PKA subunit C␣ 2 . Materials and MethodsTargeted Disruption of C␣2. C␣ 2 null mice were generated from the targeted disruption of exon 1b of the C␣ gene by homologous recombination in embryonic stem (ES) cells. The targeting vector was constructed from a 13.2-kb genomic fragment containing exons 1-3 of C␣. A loxP-flanked neomycin phosphotransferase (NEO) cassette and a mutation of the translational initiation codon were inserted into exon 1b. The linearized targeting vector was electroporated into ES cells derived from 129SV͞J mice as described (11). Germ-line chimeras were bred to C57BL͞6 mice. Pups carrying the NEO were crossed to heterozygous ROSA26-Cre recombinase transgenic mice (from P. Sorriano, Fred Hutchinson Cancer Rese...

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI,B) is essential. Mice carrying a null mutation in the gene encoding RI.8 were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CAl synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RIp mutant mice. Despite a compensatory increase in the related RIa protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RID. Since the late phase of CAl LTP also requires PKA but is normal in RI,8 mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.Little is known about the molecular mechanisms underlying homosynaptic long-term depression (LTD), an electrophysiological phenomenon thought to reflect some of the biochemical processes used in mammalian learning (1, 2). Like longterm potentiation (LTP), LTD requires an increase in intraneuronal calcium (3-5). However, in LTD a low level of calcium influx is believed to preferentially activate phosphatases (6-10), whereas in LTP a larger influx of calcium is thought to activate kinases, including (indirectly) the cAMPdependent protein kinase (PKA) w_hich is critical for the late phase of LTP (11)(12)(13)(14).In addition to mediating use-dependent changes in synaptic efficacy, PKA may be important for certain forms of learning and is specifically involved in the switch from short-to long-term memory (15). Si-nce several isoforms of PKA exist, its involvement in synaptic plasticity and learning raises a question: Do different types of learning-related neuronal changes require specific regulatory (R) or catalytic (C) subunits? In the mouse, there are four R subunits (RIa, RIP3, RIIa, RIIP) that bind cAMP and two C subunits (Ca, C13) that phosphorylate substrate proteins when released from the R subunits upon cAMP binding. In the nervous system, RI13 appears to be specific to neurons (16) and is expressed in many regions including the neocortex, the pyramidal layer of the hippocampus, and the Purkinje and granular layers of the cerebellum (17). To elucidate the role of PKA in synaptic plasticity and to determine whether specific subunits serve unique intracellular signaling functions, we used homologous recombination in embryonic stem cells to generate mice carrying a null mutation in RIP3. MATERIALS AND METHODSGeneration of Mutant Mice. The targeting of embryonic stem cells and the establishment ...

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.