Evidence of elevated intracellular calcium levels in weaver homozygote mice

In weaver (wv) mice, the substitution of a serine for a glycine (G156S) (Patil et al. 1995) alters the putative pore-forming H5 region (MacKinnon & Yellen, 1990) of the inwardly rectifying GIRK2 potassium channel and results in the loss of selectivity for K+ ions, allowing Na+ and Ca2+ ions to permeate into cells (Kofuji et al. 1996; Navarro et al. 1996; Silverman et al. 1996; Slesinger et al. 1996; Tong et al. 1996). The wv mutation results in cell death in the central nervous system and testes producing a range of neurological dysfunctions and infertility in the male (Vogelweid et al. 1993; Patil et al. 1995; Kofuji et al. 1996; Liao et al. 1996; Navarro et al. 1996; Silverman et al. 1996; Slesinger et al. 1996; Surmeier et al. 1996; Tong et al. 1996). In particular, the wv mutation affects two regions of the CNS. In cerebellum, granule cell neurons die soon after birth as they fail to differentiate and migrate into the internal granule cell layer (Goldowitz, 1989; Hess, 1996). Large numbers of granule cells are lost (Rakic & Sidman, 1973a,b; Sidman, 1976) as well as lesser numbers of Purkinje cells (Smeyne & Goldowitz, 1990) producing the prominent ataxia characteristic of these animals. In addition, there is a severe depletion of tyrosine hydroxylase positive neurons in the midbrain (substantia nigra, pars compacta) (Schmidt et al. 1982; Roffler-Tarlov & Graybiel, 1986; Triarhou et al. 1986). These anatomical changes coexist with an altered distribution of GIRK1 and GIRK2 K+ channel proteins throughout the brain (Liao et al. 1996). Putative GIRK2wv channels have been expressed in Xenopus oocytes and in CHO cells (Patil et al. 1995; Kofuji et al. 1996; Liao et al. 1996; Navarro et al. 1996; Silverman et al. 1996; Surmeier et al. 1996; Tong et al. 1996; Slesinger et al. 1996, 1997; Lauritzen et al. 1997). GIRK1 subunits expressed by themselves do not form functional channels although it is believed that GIRK2 subunits can form functional homomeric channels (assuming there was no complementation by endogenous subunits in these experiments). Homomeric GIRK2wv channels expressed in oocytes produce a constitutively active non-selective inward current (Kofuji et al. 1996; Navarro et al. 1996; Silverman et al. 1996; Slesinger et al. 1996; Tong et al. 1996) suggesting that GIRK2wv channels lose their selectivity for K+ ions and their regulation by G-proteins. Native channels are believed to be comprised of both GIRK1 and GIRK2. Co-expression of wild-type GIRK1 and GIRK2 subunits produce large K+-selective currents that are G-protein regulated (Slesinger et al. 1996). Co-expression of GIRK1 and GIRK2wv results in much smaller currents that are non-selective (Kofuji et al. 1996; Navarro et al. 1996; Slesinger et al. 1996) and which may (Slesinger et al. 1996) or may not (Kofuji et al. 1996; Navarro et al. 1996) show some G-protein regulation. In contrast to studies on expression systems, the causes leading to cell death in wv cerebellar neurons are still in dispute, but may be due to a constitutive Na+ or Ca2+ leak into neurons through GIRKwv channels (Kofuji et al. 1996; Silverman et al. 1996; Slesinger et al. 1997). In support of these conclusions, cerebellar neurons are rescued by blocking GIRK2wv channels with QX-314, MK-801, verapamil (Kofuji et al. 1996) or the Ca2+ chelator BAPTA AM (Liesi et al. 1997); GIRK2 knockout mice did not exhibit augmented neuronal cell death (Signorini et al. 1997). In contrast, Surmeier et al. (1996) proposed that cell death resulted from the loss of GIRK function. GIRK channels constitute an important inhibitory pathway in the CNS; loss of this pathway would lead to hyperexcitability and cell death. Greatly diminished GIRK currents in weaver animals have been reported by other labs as well (Lauritzen et al. 1997). Interestingly, granule cells were rescued when NR1 NMDA subunits were knocked out in weaver NR1 double mutants (Jensen et al. 1999). Thus, the exact cause of cell death remains unknown. To help clarify the cause of cerebellar granule cell death, we focused our studies on whether alterations in resting [Ca2+]i levels or changes in [Ca2+]i levels in response to elevations of external K+ could be observed in wv/wv cerebellar granule cells. In addition, if a chronic leak through GIRK2wv channels initiates a cascade leading to death, then blockade of the channels should rescue the neurons. We found that (1) resting [Ca2+]i was dramatically elevated in wv/wv cerebellar granule cells relative to +/+ neurons, (2) high-K+ stimulation produced much smaller changes in [Ca2+]i in wv/wv neurons than in +/+ neurons, and (3) the cationic channel blocker, QX-314, rescued wv/wv neurons from cell death. Our results suggest that K+ permeability is altered in wv/wv animals and are most consistent with the hypothesis that a constitutive Ca2+ and Na+ leak through GIRKwv channels probably accounts for the cerebellar granule cell death observed in wv/wv animals.